Mice that make vl binding proteins

ABSTRACT

Genetically modified mice and methods for making an using them are provided, wherein the mice comprise a replacement of all or substantially all immunoglobulin heavy chain V gene segments, D gene segments, and J gene segments with at least one light chain V gene segment and at least one light chain J gene segment. Mice that make binding proteins that comprise a light chain variable domain operably linked to a heavy chain constant region are provided. Binding proteins that contain an immunoglobulin light chain variable domain, including a somatically hypermutated light chain variable domain, fused with a heavy chain constant region, are provided. Modified cells, embryos, and mice that encode sequences for making the binding proteins are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC §119(e) of U.S.Provisional Application Ser. No. 61/369,909, filed 2 Aug. 2010, whichapplication is hereby incorporated by reference.

FIELD OF INVENTION

Immunoglobulin-like binding proteins comprising an immunoglobulin heavychain constant region fused with an immunoglobulin light chain variabledomain are provided, as well as binding proteins having animmunoglobulin light chain variable domain fused to a light chainconstant domain and an immunoglobulin light chain variable domain fusedto a heavy chain constant domain. Cells expressing such bindingproteins, mice that make them, and related methods and compositions arealso provided.

BACKGROUND

Antibodies typically comprise a tetrameric structure having twoidentical heavy chains that each comprise a heavy chain constant region(C_(H)) fused with a heavy chain variable domain (V_(H)) associated witha light chain constant region (C_(L)) fused with a light chain variabledomain (V_(L)). For a typical human IgG, an antibody molecule isapproximately about 150 kDa to about 170 kDa in size (e.g., for IgG3,which comprises a longer hinge region), depending on the subclass of IgG(e.g., IgG1, IgG3, IgG4) and (varying) length of the variable region.

In a typical antibody, V_(H) and V_(L) domains associate to form abinding site that binds a target antigen. Characteristics of theantibody with respect to affinity and specificity therefore can dependin large part on characteristics of the V_(H) and V_(L) domains. Intypical antibodies in humans and in mice, V_(H) domains couple witheither λ or κ V_(L) domains. It is also known, however, that V_(L)domains can be made that specifically bind a target antigen in theabsence of a cognate V_(H) domain (e.g., a V_(H) domain that naturallyexpresses in the context of an antibody and is associated with theparticular V_(L) domain), and that V_(H) domains can be isolated thatspecifically bind a target antigen in the absence of a cognate V_(L)domain. Thus, useful diversity in immunoglobulin-based binding proteinsis generally conferred by recombination leading to a particular V_(H) orV_(L) (and somatic hypermutation, to the extent that it occurs), as wellas by combination of a cognate V_(H)/V_(L) pair. It would be useful todevelop compositions and methods to exploit other sources of diversity.

There is a need in the art for binding proteins based on immunoglobulinstructures, including immunoglobulin variable regions such as lightchain variable regions, and including binding proteins that exhibitenhanced diversity over traditional antibodies. There is also a need forfurther methods and animals for making useful binding proteins,including binding proteins that comprise diverse light chainimmunoglobulin variable region sequences. Also in need are usefulformats for immunoglobulin-based binding proteins that provide anenhanced diversity of binding proteins from which to choose, andenhanced diversity of immunoglobulin variable domains, includingcompositions and methods for generating somatically mutated and clonallyselected immunoglobulin variable domains for use, e.g., in making humantherapeutics.

SUMMARY

In one aspect, binding proteins are described that compriseimmunoglobulin variable domains that are derived from light chain (i.e.,kappa (κ) and/or lambda (λ)) immunoglobulin variable domains, but notfrom full-length heavy chain immunoglobulin variable domains. Methodsand compositions for making binding proteins, including geneticallymodified mice, are also provided.

In one aspect, nucleic acids constructs, cells, embryos, mice, andmethods are provided for making proteins that comprise one or more κand/or λ light chain variable region immunoglobulin sequences and animmunoglobulin heavy chain constant region sequence, including proteinsthat comprise a human λ or κ light chain variable domain and a human ormouse heavy chain constant region sequence.

In one aspect, a mouse is provided, comprising an immunoglobulin heavychain locus comprising a replacement of one or more immunoglobulin heavychain variable region (V_(H)) gene segments, heavy chain diversity(D_(H)) gene segments, and heavy chain joining (J_(H)) gene segments atan endogenous mouse immunoglobulin heavy chain locus with one or morelight chain variable region (V_(L)) gene segments and one or more lightchain joining region (J_(L)) gene segments.

In one aspect, a mouse is provided, comprising an immunoglobulin heavychain locus that comprises a replacement of all or substantially allV_(H), D_(H), and J_(H) gene segments with one or more V_(L) genesegments and one or more J_(L) gene segments to form a V_(L) genesegment sequence at an endogenous heavy chain locus (VL_(H) locus),wherein the VL_(H) locus is capable of recombining with an endogenousmouse C_(H) gene to form a rearranged gene that is derived from a V_(L)gene segment, a J_(L) gene segment, and an endogenous mouse C_(H) gene.

In one embodiment, the V_(L) segments are human V_(L). In oneembodiment, the segments are human J_(L). In a specific embodiment, theV_(L) and J_(L) segments are human V_(L) and human J_(L) segments.

In one embodiment, all or substantially all V_(H), D_(H), and J_(H) genesegments are replaced with at least six human Vκ gene segments and atleast one Jκ gene segment. In one embodiment, all or substantially allV_(H), D_(H), and J_(H) gene segments are replaced with at least 16human Vκ gene segments (human Vκ) and at least one Jκ gene segment. Inone embodiment, all or substantially all V_(H), D_(H), and J_(H) genesegments are replaced with at least 30 human Vκ gene segments and atleast one Jκ gene segment. In one embodiment, all or substantially allV_(H), D_(H), and J_(H) gene segments are replaced with at least 40human Vκ gene segments and at least one Jκ gene segment. In oneembodiment, the at least one Jκ gene segment comprises two, three, four,or five human Jκ gene segments.

In one embodiment, the V_(L) segments are human Vκ segments. In oneembodiment, the human Vκ segments comprise 4-1, 5-2, 7-3, 2-4, 1-5, and1-6. In one embodiment, the κ V_(L) comprise 3-7, 1-8, 1-9, 2-10, 3-11,1-12, 1-13, 2-14, 3-15, 1-16. In one embodiment, the human Vκ segmentscomprise 1-17, 2-18, 2-19, 3-20, 6-21, 1-22, 1-23, 2-24, 3-25, 2-26,1-27, 2-28, 2-29, and 2-30. In one embodiment, the human Vκ segmentscomprise 3-31, 1-32, 1-33, 3-34, 1-35, 2-36, 1-37, 2-38, 1-39, and 2-40.

In one embodiment, the V_(L) segments are human Vκ segments and comprise4-1, 5-2, 7-3, 2-4, 1-5, 1-6, 3-7, 1-8, 1-9, 2-10, 3-11, 1-12, 1-13,2-14, 3-15, and 1-16. In one embodiment, the Vκ segments furthercomprise 1-17, 2-18, 2-19, 3-20, 6-21, 1-22, 1-23, 2-24, 3-25, 2-26,1-27, 2-28, 2-29, and 2-30. In one embodiment, the Vκ segments furthercomprise 3-31, 1-32, 1-33, 3-34, 1-35, 2-36, 1-37, 2-38, 1-39, and 2-40.

In one embodiment, the V_(L) segments are human Vλ segments and comprisea fragment of cluster A of the human light chain locus. In a specificembodiment, the fragment of cluster A of the human λ light chain locusextends from hVλ3-27 through hVλ3-1.

In one embodiment, the V_(L) segments comprise a fragment of cluster Bof the human λ light chain locus. In a specific embodiment, the fragmentof cluster B of the human λ light chain locus extends from hVλ5-52through hVλ1-40.

In one embodiment, the V_(L) segments comprise a human λ light chainvariable region sequence that comprises a genomic fragment of cluster Aand a genomic fragment of cluster B. In a one embodiment, the human λlight chain variable region sequence comprises at least one gene segmentof cluster A and at least one gene segment of cluster B.

In one embodiment, the V_(L) segments comprise at least one gene segmentof cluster B and at least one gene segment of cluster C.

In one embodiment, the V_(L) segments comprise hVλ 3-1, 4-3, 2-8, 3-9,3-10, 2-11, and 3-12. In a specific embodiment, the V_(L) segmentscomprise a contiguous sequence of the human λ light chain locus thatspans from Vλ3-12 to Vλ3-1. In one embodiment, the contiguous sequencecomprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hVλs. In aspecific embodiment, the hVλs include 3-1, 4-3, 2-8, 3-9, 3-10, 2-11,and 3-12. In a specific embodiment, the hVλs comprises a contiguoussequence of the human λ locus that spans from Vλ3-12 to Vλ3-1.

In one embodiment, the hVλs comprises 13 to 28 or more hVλs. In aspecific embodiment, the hVλs include 2-14, 3-16, 2-18, 3-19, 3-21,3-22, 2-23, 3-25, and 3-27. In a specific embodiment, the hVλs comprisea contiguous sequence of the human λ locus that spans from Vλ3-27 toVλ3-1.

In one embodiment, the V_(L) segments comprise 29 to 40 hVλs. In aspecific embodiment, the V_(L) segments comprise a contiguous sequenceof the human λ locus that spans from Vλ3-29 to Vλ3-1, and a contiguoussequence of the human λ locus that spans from Vλ5-52 to Vλ1-40. In aspecific embodiment, all or substantially all sequence between hVλ1-40and hVλ3-29 in the genetically modified mouse consists essentially of ahuman λ sequence of approximately 959 bp found in nature (e.g., in thehuman population) downstream of the hVλ1-40 gene segment (downstream ofthe 3′ untranslated portion), a restriction enzyme site (e.g., PI-SceI),followed by a human λ sequence of approximately 3,431 bp upstream of thehVλ3-29 gene segment found in nature.

In one embodiment, the Jκ is human and is selected from the groupconsisting of Jκ2, Jκ3, Jκ4, Jκ5, and a combination thereof. In aspecific embodiment, the Jκ comprises Jκ1 through Jκ5.

In one embodiment, the V_(L) segments are human Vλ segments, and the Jκgene segment comprises an RSS having a 12-mer spacer, wherein the RSS isjuxtaposed at the upstream end of the Jκ gene segment. In oneembodiment, the V_(L) gene segments are human Vλ and the VL_(H) locuscomprises two or more Jκ gene segments, each comprising an RSS having a12-mer spacer wherein the RSS is juxtaposed at the upstream end of eachJκ gene segment.

In a specific embodiment, the V_(L) segments comprise contiguous human κgene segments spanning the human κ locus from Vκ4-1 through Vκ2-40, andthe J_(L) segments comprise contiguous gene segments spanning the humanκ locus from Jκ1 through Jκ5.

In one embodiment, where the V_(L) segments are Vλ segments and no D_(H)segment is present between the V_(L) segments and J segments, the V_(L)segments are flanked downstream (i.e., juxtaposed on the downstreamside) with 23-mer RSS, and Jκ segments if present or Jλ segments ifpresent are flanked upstream (i.e., juxtaposed on the upstream side)with 12-mer RSS.

In one embodiment, where the V gene segments are Vκ gene segments and noD_(H) gene segment is present between the V gene segments and J genesegments, the Vκ gene segments are each juxtaposed on the downstreamside with a 12-mer RSS, and Jκ segments if present or Jλ segments ifpresent are each juxtaposed on the upstream side with a 23-mer RSS.

In one embodiment, the mouse comprises a rearranged gene that is derivedfrom a V_(L) gene segment, a J_(L) gene segment, and an endogenous mouseC_(H) gene. In one embodiment, the rearranged gene is somaticallymutated. In one embodiment, the rearranged gene comprises 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more N additions. In one embodiment, the N additionsand/or the somatic mutations observed in the rearranged gene derivedfrom the V_(L) segment and the J_(L) segment are 1.5-fold, 2-fold,2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or at least 5-fold morethan the number of N additions and/or somatic mutations observed in arearranged light chain variable domain (derived from the same V_(L) genesegment and the same J_(L) gene segment) that is rearranged at anendogenous light chain locus. In one embodiment, the rearranged gene isin a B cell that specifically binds an antigen of interest, wherein theB cell binds the antigen of interest with a K_(D) in the low nanomolarrange or lower (e.g., a K_(D) of 10 nanomolar or lower). In a specificembodiment, the V_(L) segment, the J_(L) segment, or both, are humangene segments. In a specific embodiment, the V_(L) and J_(L) segmentsare human κ gene segments. In one embodiment, the mouse C_(H) gene isselected from IgM, IgD, IgG, IgA and IgE. In a specific embodiment, themouse IgG is selected from IgG1, IgG2A, IgG2B, IgG2C and IgG3. Inanother specific embodiment, the mouse IgG is IgG1.

In one embodiment, the mouse comprises a B cell, wherein the B cellmakes from a locus on a chromosome of the B cell a binding proteinconsisting essentially of four polypeptide chains, wherein the fourpolypeptide chains consist essentially of (a) two identical polypeptidesthat comprise an endogenous mouse C_(H) region fused with a V_(L); and,(b) two identical polypeptides that comprise an endogenous mouse C_(L)region fused with a V_(L) region that is cognate with respect to theV_(L) region that is fused with the mouse C_(H) region, and, in oneembodiment, is a human (e.g., a human κ) V_(L) region. In oneembodiment, the V_(L) region fused to the endogenous mouse C_(H) regionis a human V_(L) region. In a specific embodiment, the human V_(L)region fused with the mouse C_(H) region is a Vκ region. In a specificembodiment, the human V_(L) region fused with the mouse C_(H) region isidentical to a V region encoded by a rearranged human germline lightchain nucleotide sequence. In a specific embodiment, the human V_(L)region fused to the mouse C_(H) region comprises two, three, four, five,six, or more somatic hypermutations. In one embodiment, the human V_(L)region fused to the mouse C_(H) region is encoded by a rearranged genethat comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

In one embodiment, at least 50% of all IgG molecules made by the mousecomprise a polypeptide that comprises an IgG isotype C_(H) region and aV_(L) region, wherein the length of said polypeptide is no longer than535, 530, 525, 520, or 515 amino acids. In one embodiment, at least 75%of all IgG molecules comprise the polypeptide recited in this paragraph.In one embodiment, at least 80%, 85%, 90%, or 95% of all IgG moleculescomprise the polypeptide recited in this paragraph. In a specificembodiment, all IgG molecules made by the mouse comprise a polypeptidethat is no longer than the length of the polypeptide recited in thisparagraph.

In one embodiment, the mouse makes a binding protein comprising a firstpolypeptide that comprises an endogenous mouse C_(H) region fused with avariable domain encoded by a rearranged human V gene segment and a Jgene segment but not a D_(H) gene segment, and a second polypeptide thatcomprises an endogenous mouse C_(L) region fused with a V domain encodedby a rearranged human V gene segment and a J gene segment but not aD_(H) gene segment, and the binding protein specifically binds anantigen with an affinity in the micromolar, nanomolar, or picomolarrange. In one embodiment, the J segment is a human J segment (e.g., ahuman κ gene segment). In one embodiment, the human V segment is a humanVκ segment. In one embodiment, the variable domain that is fused withthe endogenous mouse C_(H) region comprises a greater number of somatichypermutations than the variable region that is fused with theendogenous mouse C_(L) region; in a specific embodiment, the variableregion fused with the endogenous mouse C_(H) region comprises about 1.5,2-, 3-, 4-, or 5-fold or more somatic hypermutations than the V regionfused to the endogenous mouse C_(L) region; in a specific embodiment,the V region fused with the mouse C_(H) region comprises at least 6, 7,8, 9, 10, 11, 12, 13, 14, or 15 or more somatic hypermutations than theV region fused with the mouse C_(L) region. In one embodiment, the Vregion fused with the mouse C_(H) region is encoded by a rearranged genethat comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

In one embodiment, the mouse expresses a binding protein comprising afirst light chain variable domain (V_(L)1) fused with an immunoglobulinheavy chain constant region sequence and a second light chain variabledomain (V_(L)2) fused with an immunoglobulin light chain constantregion, wherein V_(L)1 comprises a number of somatic hypermutations thatis about 1.5- to about 5-fold higher or more than the number of somatichypermutations present in V_(L)2. In one embodiment, the number ofsomatic hypermutations in V_(L)1 is about 2- to about 4-fold higher thanin V_(L)2. In one embodiment, the number of somatic hypermutations inV_(L)1 is about 2- to about 3-fold higher than in V_(L)2. In oneembodiment, V_(L)1 is encoded by a sequence that comprises 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 or more N additions.

In one aspect, a genetically modified mouse is provided that expressesan immunoglobulin that consists essentially of the followingpolypeptides: a first two identical polypeptides that each consistsessentially of a C_(H) region fused with a variable domain that isderived from gene segments that consist essentially of a V_(L) genesegment and a J_(L) gene segment, and a second two identicalpolypeptides that each consists essentially of a C_(L) region fused witha variable domain that is derived from gene segments that consistessentially of a V_(L) segment and a J_(L) segment.

In a specific embodiment, the two identical polypeptides that have theC_(H) region have a mouse C_(H) region.

In a specific embodiment, the two identical polypeptides that have theC_(L) region have a mouse C_(L) region.

In one embodiment, the variable domain fused with the C_(L) region is avariable domain that is cognate with the variable domain fused to theC_(H) region.

In one embodiment, the variable domain that is fused with the endogenousmouse C_(H) region comprises a greater number of somatic hypermutationsthan the variable domain that is fused with the endogenous mouse C_(L)region; in a specific embodiment, the variable domain fused with theendogenous mouse C_(H) region comprises about 1.5-fold, 2-fold,2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold or more somatichypermutations than the variable domain fused to the endogenous mouseC_(L) region. In one embodiment, the variable domain fused with theendogenous mouse C_(L) region is encoded by a gene that comprises 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more N additions.

In one embodiment, one or more of the V segments and the J segments arehuman gene segments. In a specific embodiment, both the V segments andthe J segments are human κ gene segments. In another specificembodiment, both of the V segments and the J segments are human λ genesegments. In one embodiment, the V segments and the J segments areindependently selected from human κ and human λ gene segments. In aspecific embodiment, the V segments are Vκ segments and the J segmentsare Jλ segments. In another specific embodiment, the V segments are Vλsegments and the J segments are Jκ segments.

In one embodiment, one or more of the variable domains fused with theC_(L) region and the variable domains fused with the C_(H) region arehuman variable domains. In a specific embodiment, the human variabledomains are human Vκ domains. In another specific embodiment, the humanvariable domains are Vλ domains. In one embodiment, the human domainsare independently selected from human Vκ and human Vλ domains. In aspecific embodiment, the human variable domain fused with the C_(L)region is a human Vλ domain and the human variable domain fused with theC_(H) region is a human Vκ domain. In another embodiment, the humanvariable domain fused with the C_(L) region is a human Vκ domain and thehuman variable domain fused with the C_(H) is a human Vλ domain.

In one embodiment, the V_(L) gene segment of the first two identicalpolypeptides is selected from a human Vλ segment and a human Vκ segment.In one embodiment, the V_(L) segment of the second two identicalpolypeptides is selected from a human Vλ segment and a human Vκ segment.In a specific embodiment, the V_(L) segment of the first two identicalpolypeptides is a human Vκ segment and the V_(L) segment of the secondtwo identical polypeptides is selected from a human Vκ segment and ahuman Vλ segment. In a specific embodiment, the V_(L) segment of thefirst two identical polypeptides is a human Vλ segment and the V_(L)segment of the second two identical polypeptides is selected from ahuman Vλ segment and a human Vκ segment. In a specific embodiment, thehuman V_(L) segment of the first two identical polypeptides is a humanVκ segment, and the human V_(L) segment of the second two identicalpolypeptides is a human Vκ segment.

In one embodiment, the IgG of the mouse comprises a binding protein madein response to an antigen, wherein the binding protein comprises apolypeptide that consists essentially of a variable domain and a C_(H)region, wherein the variable domain is encoded by a nucleotide sequencethat consists essentially of a rearranged V_(L) segment and a rearrangedJ segment, and wherein the binding protein specifically binds an epitopeof the antigen with a K_(D) in the micromolar, nanomolar, or picomolarrange.

In one aspect, a mouse is provided, wherein all or substantially all ofthe IgG made by the mouse in response to an antigen comprises a heavychain that comprises a variable domain, wherein the variable domain isencoded by a rearranged gene derived from gene segments that consistessentially of a V gene segment and a J gene segment. In one embodiment,the rearranged gene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more Nadditions.

In one embodiment, the V segment is a V segment of a light chain. In oneembodiment, the light chain is selected from a κ light chain and a λlight chain. In a specific embodiment, the light chain is a κ lightchain. In a specific embodiment, the V segment is a human V segment. Ina specific embodiment, the V segment is a human Vκ segment and the Jsegment is a human Jκ segment.

In one embodiment, the J segment is a J segment of a light chain. In oneembodiment, the light chain is selected from a κ light chain and a λlight chain. In a specific embodiment, the light chain is a κ lightchain. In a specific embodiment, the J segment is a human J segment. Inanother embodiment, the J segment is a J segment of a heavy chain (i.e.,a J_(H)). In a specific embodiment, the heavy chain is of mouse origin.In another specific embodiment, the heavy chain is of human origin.

In one embodiment, the variable domain of the heavy chain that is madefrom no more than a V segment and a J segment is a somatically mutatedvariable domain.

In one embodiment, the variable domain of the heavy chain that is madefrom no more than a V segment and a J segment is fused to a mouse C_(H)region.

In a specific embodiment, all or substantially all of the IgG made bythe mouse in response to an antigen comprises a variable domain that isderived from no more than one human V segment and no more than one humanJ segment, and the variable domain is fused to a mouse IgG constantregion, and the IgG further comprises a light chain that comprises ahuman V_(L) domain fused with a mouse C_(L) region. In a specificembodiment, the V_(L) domain fused with the mouse C_(L) region isderived from a human Vκ segment and a human Jκ segment. In a specificembodiment, the V_(L) domain fused with the mouse C_(L) region isderived from a human Vλ segment and a human Jλ segment.

In one aspect, a mouse is provided that makes an IgG comprising a firstCDR3 on a polypeptide comprising a C_(H) region and a second CDR3 on apolypeptide comprising a C_(L) region, wherein both the first CDR3 andthe second CDR3 are each independently derived from no more than twogene segments, wherein the two gene segments consist essentially of aV_(L) gene segment and a J_(L) gene segment. In one embodiment, the CDR3on the polypeptide comprising the C_(H) region comprises a sequence thatis derived from a CDR3 nucleotide sequence that comprises 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more N additions.

In one embodiment, the V_(L) segment and the J_(L) segment are humangene segments. In one embodiment, the V_(L) segment and the J_(L)segment are κ gene segments. In one embodiment, the V_(L) segment andthe J_(L) segment are λ gene segments.

In one aspect, a mouse is provided that makes an IgG comprising a firstCDR3 on a first polypeptide comprising a C_(H) region and a second CDR3on a second polypeptide comprising a C_(L) region, wherein both thefirst CDR3 and the second CDR3 each comprise a sequence of amino acidswherein more than 75% of the amino acids are derived from a V genesegment. In one embodiment, the CDR3 on the polypeptide comprising theC_(H) region comprises a sequence that is derived from a CDR3 nucleotidesequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more Nadditions.

In one embodiment, more than 80%, more than 90%, or more than 95% of theamino acids of the first CDR3, and more than 80%, more than 90%, or morethan 95% of the amino acids of the second CDR3, are derived from a lightchain V segment.

In one embodiment, no more than two amino acids of the first CDR3 arederived from a gene segment other than a light chain V segment. In oneembodiment, no more than two amino acids of the second CDR3 are derivedfrom a gene segment other than a light chain V segment. In a specificembodiment, no more than two amino acids of the first CDR3 and no morethan two amino acids of the second CDR3 are derived from a gene segmentother than a light chain V segment. In one embodiment, no CDR3 of theIgG comprises an amino acid sequence derived from a D gene segment. Inone embodiment, the CDR3 of the first polypeptide does not comprise asequence derived from a D segment.

In one embodiment, the V segment is a human V gene segment. In aspecific embodiment, the V segment is a human Vκ gene segment.

In one embodiment, the first and/or the second CDR3 have at least one,two, three, four, five, or six somatic hypermutations. In oneembodiment, the first CDR3 is encoded by a nucleic acid sequence thatcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

In one embodiment, the first CDR3 consists essentially of amino acidsderived from a human light chain V gene segment and a human light chainJ gene segment, and the second CDR3 consists essentially of amino acidsderived from a human light chain V gene segment and a human light chainJ gene segment. In one embodiment, the first CDR3 is derived from anucleic acid sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore N additions. In one embodiment, the first CDR3 is derived from nomore than two gene segments, wherein the no more than two gene segmentsare a human Vκ gene segment and a human Jκ gene segment; and the secondCDR3 is derived from no more than two gene segments, wherein the no morethan two gene segments are a human Vκ gene segment and a J gene segmentselected from a human Jκ segment, a human Jλ segment, and a human J_(H)segment. In one embodiment, the first CDR3 is derived from no more thantwo gene segments, wherein the no more than two gene segments are ahuman Vλ segment and a J segment selected from a human Jκ segment, ahuman Jλ segment, and a human J_(H) segment.

In one aspect, a mouse is provided that makes an IgG that does notcontain an amino acid sequence derived from a D_(H) gene segment,wherein the IgG comprises a first polypeptide having a first V_(L)domain fused with a mouse C_(L) region and a second polypeptide having asecond V_(L) domain fused with a mouse C_(H) region, wherein the firstV_(L) domain and the second V_(L) domain are not identical. In oneembodiment, the first and second V_(L) domains are derived fromdifferent V segments. In another embodiment, the first and second V_(L)domains are derived from different J segments. In one embodiment, thefirst and second V_(L) domains are derived from identical V and Jsegments, wherein the second V_(L) domain comprises a higher number ofsomatic hypermutations as compared to the first V_(L) domain.

In one embodiment, the first and the second V_(L) domains areindependently selected from human and mouse V_(L) domains. In oneembodiment, the first and second V_(L) domains are independentlyselected from Vκ and Vλ domains. In a specific embodiment, the firstV_(L) domain is selected from a Vκ domain and a Vλ domain, and thesecond V_(L) domain is a Vκ domain. In another specific embodiment, theVκ domain is a human Vκ domain.

In one aspect, a mouse is provided, wherein all or substantially all ofthe IgG made by the mouse consists essentially of a light chain having afirst human V_(L) domain fused with a mouse C_(L) domain, and a heavychain having a second human V_(L) domain fused with a mouse C_(H)domain.

In one embodiment, the human V_(L) domain fused with the mouse C_(H)domain is a human Vκ domain.

In one embodiment, the first and the second human V_(L) domains are notidentical.

In one aspect, a mouse is provided, wherein at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or about 100% of the immunoglobulin G madeby the mouse consists essentially of a dimer of (a) a first polypeptidethat consists essentially of an immunoglobulin V_(L) domain and animmunoglobulin C_(L) region; and, (b) a second polypeptide of no morethan 535 amino acids in length, wherein the second polypeptide consistsessentially of a C_(H) region and a V domain that lacks a sequencederived from a D_(H) gene segment.

In one embodiment, the second polypeptide is about 435-535 amino acidsin length. In a specific embodiment, the second polypeptide is about435-530 amino acids in length. In a specific embodiment, the secondpolypeptide is about 435-525 amino acids in length. In a specificembodiment, the second polypeptide is about 435-520 amino acids inlength. In a specific embodiment, the second polypeptide is about435-515 amino acids in length.

In one embodiment, in about 90% or more of the IgG made by the mouse thesecond polypeptide is no more than about 535 amino acids in length.

In one embodiment, in about 50% or more of the IgG made by the mouse thesecond polypeptide is no more than about 535 amino acids in length. Inone embodiment, in about 50% or more of the immunoglobulin G made by themouse the second polypeptide is no more than about 530, 525, 520, 515,510, 505, 500, 495, 490, 485, 480, 475, 470, 465, 460, 455, or 450 aminoacids in length. In one embodiment, about 60%, 70%, 80%, 90%, or 95% ormore of the IgG made by the mouse is of the recited length. In aspecific embodiment, all or substantially all of the IgG made by themouse is of the recited length.

In one embodiment, the V domain of the second polypeptide is a V_(L)domain. In a specific embodiment, the V domain of the second polypeptideis selected from a Vκ and a Vλ domain. In a specific embodiment, the Vdomain of the second polypeptide is a human Vκ or Vλ domain.

In one aspect, a mouse is provided that expresses from a nucleotidesequence in its germline a polypeptide that comprises a light chainvariable sequence (e.g., a V and/or J sequence), a D_(H) sequence, and aheavy chain constant region.

In one embodiment, the mouse expresses the polypeptide from anendogenous mouse heavy chain locus that comprises a replacement of allor substantially all functional endogenous mouse heavy chain variablelocus gene segments with a plurality of human gene segments at theendogenous mouse heavy chain locus.

In one embodiment, the polypeptide comprises a V_(L) sequence derivedfrom a Vλ or a Vκ gene segment, the polypeptide comprises a CDR3 derivedfrom a D_(H) gene segment, and the polypeptide comprises a sequencederived from a J_(H) or Jλ or Jκ gene segment.

In one embodiment, the mouse comprises an endogenous mouse heavy chainimmunoglobulin locus comprising a replacement of all functional V_(H)gene segments with one or more human light chain Vλ gene segmentswherein the one or more human Vλ segments each have juxtaposed on thedownstream side a 23-mer spaced recombination signal sequence (RSS),wherein the Vλ segments are operably linked to a human or mouse D_(H)segment that has juxtaposed upstream and downstream a 12-mer spaced RSS;the D_(H) gene segment is operably linked with a J segment juxtaposedupstream with a 23-mer spaced RSS that is suitable for recombining withthe 12-mer spaced RSS juxtaposing the D_(H) gene segment; wherein the V,D_(H), and J segments are operably linked to a nucleic acid sequenceencoding a heavy chain constant region.

In one embodiment, the mouse comprises an endogenous mouse heavy chainimmunoglobulin locus comprising a replacement of all functional V_(H)gene segments with one or more human Vκ gene segments each juxtaposed onthe downstream side with a 12-mer spaced recombination signal sequence(RSS), wherein the V segments are operably linked to a human or mouseD_(H) segment that is juxtaposed both upstream and downstream with a23-mer spaced RSS; the D_(H) segment is operably linked with a J segmentjuxtaposed on the upstream side with a 12-mer spaced RSS that issuitable for recombining with the 23-mer spaced RSS juxtaposing theD_(H) segment; wherein the V, D_(H), and gene segments are operablylinked to a nucleic acid sequence encoding a heavy chain constantregion.

In one embodiment, the heavy chain constant region is an endogenousmouse heavy chain constant region. In one embodiment, the nucleic acidsequence encodes a sequence selected from a C_(H)1, a hinge, a C_(H)2, aC_(H)3, and a combination thereof. In one embodiment, one or more of theC_(H)1, hinge, C_(H)2, and C_(H)3 are human.

In one embodiment, the mouse comprises an endogenous mouse heavy chainimmunoglobulin locus comprising a replacement of all functional V_(H)gene segments with a plurality of human Vλ or Vκ gene segments eachjuxtaposed downstream with 23-mer spaced RSS, a plurality of human D_(H)segments juxtaposed both upstream and downstream by a 12-mer spaced RSS,a plurality of human J segments (J_(H) or Jλ or Jκ) juxtaposed bothupstream and downstream with a 23-mer spaced RSS, wherein the locuscomprises an endogenous mouse constant region sequence selected fromC_(H)1, hinge, C_(H)2, C_(H)3, and a combination thereof. In a specificembodiment, the mouse comprises all or substantially all functionalhuman Vλ or Vκ segments, all or substantially all functional human D_(H)segments, and all or substantially all J_(H) or Jλ or Jκ segments.

In one embodiment, the mouse expresses an antigen-binding proteincomprising (a) a polypeptide that comprises a human light chain sequencelinked to a heavy chain constant sequence comprising a mouse sequence;and (b) a polypeptide that comprises a human light chain variable regionlinked to a human or mouse light chain constant sequence. In a specificembodiment, the light chain sequence is a human light chain sequence,and upon exposure to a protease that is capable of cleaving an antibodyinto an Fc and a Fab, a fully human Fab is formed that comprises atleast four light chain CDRs, wherein the at least four light chain CDRsare selected from λ sequences, κ sequences, and a combination thereof.In one embodiment, the Fab comprises at least five light chain CDRs. Inone embodiment, the Fab comprises six light chain CDRs. In oneembodiment, at least one CDR of the Fab comprises a sequence derivedfrom a Vλ segment or a Vκ segment, and the at least one CDR furthercomprises a sequence derived from a D segment. In one embodiment, the atleast one CDR is a CDR3 and the CDR is derived from a human Vκ segment,a human D segment, and a human Jκ segment.

In one embodiment, the polypeptide of comprises a variable regionderived from a human Vλ or Vκ gene segment, a human D_(H) gene segment,and a human J_(H) or Jλ or Jκ gene segment. In a specific embodiment,the heavy chain constant sequence is derived from a human C_(H)1 and amouse C_(H)2 and a mouse C_(H)3 sequence.

In one aspect, a mouse is provided that comprises in its germline anunrearranged human Vκ or Vλ gene segment operably linked to a human Jgene segment and a heavy chain constant region sequence, wherein themouse expresses a V_(L) binding protein that comprises a human Vκ domainfused with a heavy chain constant region, and wherein the mice exhibit apopulation of splenic B cells that express V_(L) binding proteins inCD19⁺ B cells, including transitional B cells (CD19⁺IgM^(hi)IgD^(int)),and mature B cells (CD19⁺IgM^(int)IgD^(hi)).

In one aspect, a mouse is provided that comprises in its germline anunrearranged human Vκ or Vλ gene segment operably linked to a human Jgene segment and a heavy chain constant region sequence, wherein themouse expresses on a B cell an immunoglobulin that comprises a lightchain variable domain fused with a heavy chain constant region, whereinthe lymphocyte population in bone marrow of the mice exhibit a pro/pre Bcell population that is about the same in number as in a pro/pre B cellpopulation of a wild-type mouse (lymphocytes in bone marrow).

In one embodiment, the mice comprise at least 6 unrearranged hVκ genesegments and one or more unrearranged hJκ gene segments, and the micecomprise a lymphocyte-gated and IgM⁺ spleen cell population expressing aV_(L) binding protein, wherein the population is at least 75% as largeas a lymphocyte-gated and IgM⁺ spleen cell population of a wild-typemouse.

In one embodiment, the mice exhibit a mature B cell-gated (CD19⁺)splenocyte population of IgD⁺ cells and IgM⁺ cells that total about 90%;in one embodiment, the mature B cell-gated (CD19⁺) splenocyte populationof IgD⁺ cells and IgM⁺ cells of the modified mouse is about the same(e.g., within 10%, or within 5%) as the total of IgD⁺ cells and IgM⁺cells of a wild-type mouse that are mature B cell-gated (CD19⁺)splenocytes.

In one aspect, a mouse is provided that expresses an immunoglobulinprotein from a modified endogenous heavy chain locus in its germline,wherein the modified endogenous heavy chain locus lacks a functionalmouse heavy chain V gene segment and the locus comprises unrearrangedlight chain V gene segments and unrearranged J gene segments, whereinthe unrearranged light chain V gene segments and unrearranged J genesegments are operably linked with a heavy chain constant regionsequence; wherein the immunoglobulin protein consists essentially of afirst polypeptide and a second polypeptide, wherein the firstpolypeptide comprises an immunoglobulin light chain sequence and animmunoglobulin heavy chain constant sequence, and the second polypeptidecomprises an immunoglobulin light chain variable domain and a lightchain constant region.

In one aspect, a mouse is provided that expresses an immunoglobulinprotein, wherein the immunoglobulin protein lacks a heavy chainimmunoglobulin variable domain, and the immunoglobulin protein comprisesa first variable domain derived from a light chain gene, and a secondvariable domain derived from a light chain gene, wherein the firstvariable domain and the second variable domain are cognate with respectto one another, wherein the first and the second light chain variabledomains are not identical, and wherein the first and the second lightchain variable domains associate and when associated specifically bindan antigen of interest.

In one aspect, a mouse is provided that makes from unrearranged genesegments in its germline an immunoglobulin protein comprising variableregions that are wholly derived from gene segments that consistessentially of unrearranged human gene segments, wherein theimmunoglobulin protein comprises an immunoglobulin light chain constantsequence and an immunoglobulin heavy chain constant sequence selectedfrom the group consisting of a C_(H)1, a hinge, a C_(H)2, a C_(H)3, anda combination thereof.

In one aspect, a mouse is provided that makes from unrearranged genesegments in its germline an immunoglobulin protein comprising variableregions, wherein all CDR3s of all variable regions are generatedentirely from light chain V and J gene segments, and optionally one ormore somatic hypermutations, e.g., one or more N additions.

In one aspect, a mouse is provided that makes a somatically mutatedimmunoglobulin protein derived from unrearranged human immunoglobulinlight chain variable region gene segments in the germline of the mouse,wherein the immunoglobulin protein lacks a CDR that comprises a sequencederived from a D gene segment, wherein the immunoglobulin proteincomprises a first CDR3 on a light chain variable domain fused with alight chain constant region, comprises a second CDR3 on a light chainvariable domain fused with a heavy chain constant region, and whereinthe second CDR3 is derived from a rearranged light chain variable regionsequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more Nadditions.

In one aspect, a mouse as described herein is provided, wherein themouse comprises a functionally silenced light chain locus selected froma λ locus, a κ locus, and a combination thereof. In one embodiment, themouse comprises a deletion of a λ and/or a κ locus, in whole or in part,such that the λ and/or κ locus is nonfunctional.

In one aspect, a mouse embryo is provided, comprising a cell thatcomprises a modified immunoglobulin locus as described herein. In oneembodiment, the mouse is a chimera and at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% of the cells of the embryo comprise amodified immunoglobulin locus as described herein. In one embodiment, atleast 96%. 97%, 98%, 99%, or 99.8% of the cells of the embryo comprise amodified immunoglobulin locus as described herein. In one embodiment,the embryo comprises a host cell and a cell derived from a donor EScell, wherein the cell derived from the donor ES cell comprises amodified immunoglobulin locus as described herein. In one embodiment,the embryo is a 2-, 4-, 8, 16-, 32, or 64-cell stage host embryo, or ablastocyst, and further comprises a donor ES cell comprising a modifiedimmunoglobulin locus as described herein.

In one aspect, a mouse or a cell made using a nucleic acid construct asdescribed herein is provided.

In one aspect, a mouse made using a cell as described herein isprovided. In one embodiment, the cell is a mouse ES cell.

In one aspect, use of a mouse as described herein to make a nucleic acidsequence encoding a first human light chain immunoglobulin variablesequence (V_(L)1) that is cognate with a second human light chainimmunoglobulin variable sequence (V_(L)2), wherein the V_(L)1 fused witha human immunoglobulin light chain constant region (polypeptide 1)expresses with V_(L)2 fused with a human immunoglobulin heavy chainconstant region (polypeptide 2), as a dimer of polypeptide 1/polypeptide2, to form a V_(L)1-V_(L)2 antibody.

In one aspect, use of a mouse as described herein to make a nucleic acidsequence encoding a human immunoglobulin light chain variable sequencethat is fused with a human immunoglobulin heavy chain sequence, whereinthe nucleic acid sequence encodes a human V_(L)-C_(H) polypeptide,wherein the human V_(L)-C_(H) polypeptide expresses as a dimer, andwherein the dimer expresses in the absence of an immunoglobulin lightchain (e.g., in the absence of a human λ or human κ light chain). In oneembodiment, the V_(L)-C_(H) dimer specifically binds an antigen ofinterest in the absence of a λ light chain and in the absence of a κlight chain.

In one aspect, use of a mouse as described herein to make a nucleic acidsequence encoding all or a portion of an immunoglobulin variable domain.In one embodiment, the immunoglobulin variable domain is a human Vλ orhuman Vκ domain.

In one aspect, use of a mouse as described herein to make a fully humanFab (comprising a first human V_(L) fused with a human light chainconstant region, and a second human V_(L) fused with a human heavy chainconstant region sequence) or a fully human F(ab)₂ is provided.

In one aspect, use of a mouse as described herein to make animmortalized cell line is provided. In one embodiment, the immortalizedcell line comprises a nucleic acid sequence encoding a human Vλ or Vκdomain operably linked to a nucleic acid sequence that comprises a mouseconstant region nucleic acid sequence.

In one aspect, use of a mouse as described herein to make a hybridoma orquadroma is provided.

In one aspect, a cell is provided, comprising a modified immunoglobulinlocus as described herein. In one embodiment, the cell is selected froma totipotent cell, a pluripotent cell, an induced pluripotent stem cell(iPS), and an ES cell. In a specific embodiment, the cell is a mousecell, e.g., a mouse ES cell. In one embodiment, the cell is homozygousfor the modified immunoglobulin locus.

In one aspect, a cell is provided, comprising a nucleic acid sequenceencoding a first polypeptide that comprises a first somatically mutatedhuman Vκ or Vλ sequence fused to a human heavy chain constant regionsequence.

In one embodiment, the cell further comprises a second polypeptide chainthat comprises a second somatically mutated human Vκ or Vλ sequencefused to a human light chain constant region sequence.

In one embodiment, the human Vκ or Vλ sequence of the first polypeptideis cognate with the human Vκ or Vλ sequence of the second polypeptide.

In one embodiment, the Vκ or Vλ of the first polypeptide and the humanVκ or Vλ of the second polypeptide when associated specifically bind anantigen of interest. In a specific embodiment, the first polypeptidecomprises a variable domain consisting essentially of a human Vκ, andthe second polypeptide comprises a variable domain consisting of a humanVκ that is cognate with the human Vκ of the first polypeptide, and thehuman constant region sequence is an IgG sequence.

In one embodiment, the cell is selected from a CHO cell, a COS cell, a293 cell, a HeLa cell, and a human retinal cell expressing a viralnucleic acid sequence (e.g., a PERC.6™ cell.

In one aspect, a somatic mouse cell is provided, comprising a chromosomethat comprises a genetic modification as described herein.

In one aspect, a mouse germ cell is provided, comprising a nucleic acidsequence that comprises a genetic modification as described herein.

In one aspect, a pluripotent, induced pluripotent, or totipotent cellderived from a mouse as described herein is provided. In a specificembodiment, the cell is a mouse embryonic stem (ES) cell.

In one aspect, use of a cell as described herein for the manufacture ofa mouse, a cell, or a therapeutic protein (e.g., an antibody or otherantigen-binding protein) is provided.

In one aspect, a nucleic acid construct is provided that comprises ahuman D_(H) gene segment juxtaposed upstream and downstream with a23-mer spaced RSS. In a specific embodiment, the nucleic acid constructcomprises a homology arm that is homologous to a human genomic sequencecomprising human Vκ gene segments. In one embodiment, the targetingconstruct comprises all or substantially all human D_(H) gene segmentseach juxtaposed upstream and downstream with a 23-mer spaced RSS.

In one aspect, a nucleic acid construct is provided that comprises ahuman Jκ gene segment juxtaposed upstream with a 12-mer spaced RSS. In aspecific embodiment, the nucleic acid construct comprises a firsthomology arm that contains homology to a human genomic D_(H) genesequence that is juxtaposed upstream and downstream with a 23-mer spacedRSS. In one embodiment, the nucleic acid construct comprises a secondhomology arm that contains homology to a human genomic J gene sequenceor that contains homology to a mouse heavy chain constant regionsequence or that contains homology to a J-C intergenic sequence upstreamof a mouse constant region heavy chain sequence.

In one aspect, a nucleic acid construct is provided that comprises ahuman Vλ segment juxtaposed downstream with a 23-mer spaced RSS, a humanD_(H) segment juxtaposed upstream and downstream with a 12-mer spacedRSS, and a human J segment selected from a Jκ segment juxtaposedupstream with a 23-mer spaced RSS, a human Jλ segment juxtaposedupstream with a 23-mer spaced RSS, and a human J_(H) segment juxtaposedupstream with a 23-mer spaced RSS. In one embodiment, the constructcomprises a homology arm that contains homology to a mouse constantregion sequence, a J-C intergenic mouse sequence, and/or a human Vλsequence.

In one embodiment, the nucleic acid construct comprises a human λ lightchain variable region sequence that comprises a fragment of cluster A ofthe human λ light chain locus. In a specific embodiment, the fragment ofcluster A of the human λ light chain locus extends from hVλ3-27 throughhVλ3-1.

In one embodiment, the nucleic acid construct comprises a human λ lightchain variable region sequence that comprises a fragment of cluster B ofthe human λ light chain locus. In a specific embodiment, the fragment ofcluster B of the human λ light chain locus extends from hVλ5-52 throughhVλ1-40.

In one embodiment, nucleic acid construct comprises a human λ lightchain variable region sequence that comprises a genomic fragment ofcluster A and a genomic fragment of cluster B. In a one embodiment, thehuman λ light chain variable region sequence comprises at least one genesegment of cluster A and at least one gene segment of cluster B.

In one embodiment, the human λ light chain variable region sequencecomprises at least one gene segment of cluster B and at least one genesegment of cluster C.

In one aspect, a nucleic acid construct is provided, comprising a humanD_(H) segment juxtaposed upstream and downstream with a 23-mer spacedRSS normally found in nature flanking either a Jκ, a J_(H), a Vλ, or aV_(H) segment. In one embodiment, the nucleic acid construct comprises afirst homology arm homologous to a human V-J intergenic region orhomologous to a human genomic sequence comprising a human V genesegment. In one embodiment, the nucleic acid construct comprises asecond homology arm homologous to a human or mouse heavy chain constantregion sequence. In a specific embodiment, the human or mouse heavychain constant region sequence is selected from a C_(H)1, hinge, C_(H)2,C_(H)3, and a combination thereof. In one embodiment, the nucleic acidconstruct comprises a human J gene segment flanked upstream with a12-mer RSS. In one embodiment, the nucleic acid construct comprises asecond homology arm that contains homology to a J gene segment flankedupstream with a 12-mer RSS. In one embodiment, the J gene segment isselected from a human Jκ, a human Jλ, and a human J_(H).

In one aspect, a nucleic acid construct is provided that comprises ahuman D_(H) segment juxtaposed upstream and downstream with a 23-merspaced RSS, and a site-specific recombinase recognition sequence, e.g.,a sequence recognized by a site-specific recombinase such as a Cre, aFlp, or a Dre protein.

In one aspect, a nucleic acid construct is provided that comprises ahuman Vλ or a human Vκ segment, a D_(H) segment juxtaposed upstream anddownstream with a 12-mer or a 23-mer spaced RSS, and a human J segmentwith a 12-mer or a 23-mer spaced RSS, wherein the 12-mer or 23-merspaced RSS is positioned immediately 5′ to the human J segment (i.e.,with respect to the direction of transcription). In one embodiment, theconstruct comprises a human Vλ juxtaposed with a 3′ 23-mer spaced RSS, ahuman D_(H) segment juxtaposed upstream and downstream with a 12-merspaced RSS, and a human Jκ segment juxtaposed with a 5′ 23-mer spacedRSS. In one embodiment, the construct comprises a human Vκ juxtaposedwith a 3′ 12-mer spaced RSS, a human D_(H) segment juxtaposed upstreamand downstream with a 23-mer spaced RSS, and a human Jλ segmentjuxtaposed with a 5′ 12-mer spaced RSS.

In one aspect, a targeting vector is provided, comprising (a) a firsttargeting arm and a second targeting arm, wherein the first and secondtargeting arms are independently selected from human and mouse targetingarms, wherein the targeting arms direct the vector to an endogenous ormodified immunoglobulin V region gene locus; and, (b) a contiguoussequence of human V_(L) gene segments or a contiguous sequence of humanV_(L) gene segments and at least one human Jκ gene segment, wherein thecontiguous sequence is selected from the group consisting of (i) hVκ4-1through hVκ1-6 and Jκ1, (ii) hVκ4-1 through hVκ1-6 and Jκ1 through Jκ2,(iii) hVκ4-1 through hVκ1-6 and Jκ1 through Jκ3, (iv) hVκ4-1 throughhVκ1-6 and Jκ1 through Jκ4, (v) hVκ4-1 through hVκ1-6 and Jκ1 throughJκ5, (vi) hVκ3-7 through hVκ1-16, (vii) hVκ1-17 through hVκ2-30, (viii)hVκ3-31 through hVκ2-40, and (ix) a combination thereof.

In one embodiment, the targeting arms that direct the vector to anendogenous or modified immunoglobulin locus are identical orsubstantially identical to a sequence at the endogenous or modifiedimmunoglobulin locus.

In one aspect, use of a nucleic acid construct as described herein forthe manufacture of a mouse, a cell, or a therapeutic protein (e.g., anantibody or other antigen-binding protein) is provided.

In one aspect, use of a nucleic acid sequence from a mouse as describedherein to make a cell line for the manufacture of a human therapeutic isprovided. In one embodiment, the human therapeutic is a binding proteincomprising a human light chain variable sequence (e.g., derived from ahuman Vλ or human Vκ segment) fused with a human heavy chain constantsequence. In one embodiment, the human therapeutic comprises a firstpolypeptide that is a human λ or κ immunoglobulin light chain, and asecond polypeptide that comprises a human Vλ or human Vκ variablesequence fused with a human heavy chain constant sequence.

In one aspect, an expression system is provided, comprising a mammaliancell transfected with a DNA construct that encodes a polypeptide thatcomprises a somatically mutated human V_(L) domain fused with a humanC_(H) domain.

In one embodiment, the expression system further comprises a nucleotidesequence that encodes an immunoglobulin V_(L) domain fused with a humanC_(L) domain, wherein the V_(L) domain fused with the human C_(L) domainis a cognate light chain with the V_(L) domain fused with the humanC_(H) domain.

In one embodiment, the mammalian cell is selected from a CHO cell, a COScell, a Vero cell, a 293 cell, and a retinal cell that expresses a viralgene (e.g., a PER.C6™ cell).

In one aspect, a method for making a binding protein is provided,comprising obtaining a nucleotide sequence encoding a V_(L) domain froma gene encoding a V_(L) region fused to a C_(H) region from a cell of amouse as described herein, and cloning the nucleotide sequence encodingthe V_(L) region sequence in frame with a gene encoding a human C_(H)region to form a human binding protein sequence, expressing the humanbinding protein sequence in a suitable cell.

In one embodiment, the mouse has been immunized with an antigen ofinterest, and the V_(L) region fused to the C_(H) region specificallybinds (e.g., with a K_(D) in the micromolar, nanomolar, or picomolarrange) an epitope of the antigen of interest. In one embodiment,nucleotide sequence encoding the V_(L) region fused to the C_(H) regionis somatically mutated in the mouse.

In one embodiment, the suitable cell is selected from a B cell, ahybridoma, a quadroma, a CHO cell, a COS cell, a 293 cell, a HeLa cell,and a human retinal cell expressing a viral nucleic acid sequence (e.g.,a PERC.6™ cell).

In one embodiment, the C_(H) region comprises a human IgG isotype. In aspecific embodiment, the human IgG is selected from an IgG1, IgG2, andIgG4. In another specific embodiment, the human IgG is IgG1. In anotherspecific embodiment, the human IgG is IgG4. In another specificembodiment, the human IgG4 is a modified IgG4. In one embodiment, themodified IgG4 comprises a substitution in the hinge region. In aspecific embodiment, the modified IgG4 comprises a substitution at aminoacid residue 228 relative to a wild-type human IgG4, numbered accordingto the EU numbering index of Kabat. In a specific embodiment, thesubstitution at amino acid residue 228 is a S228P substitution, numberedaccording to the EU numbering index of Kabat.

In one embodiment, the cell further comprises a nucleotide sequenceencoding a V_(L) domain from a light chain that is cognate to the V_(L)domain fused to the C_(H) region, and the method further comprisesexpressing the nucleotide sequence encoding the cognate V_(L) domainfused to a human Cκ or Cλ domain.

In one aspect, a method for making a genetically modified mouse isprovided, comprising replacing at an endogenous mouse heavy chain locusone or more immunoglobulin heavy chain gene segments of a mouse with oneor more human immunoglobulin light chain gene segments. In oneembodiment, the replacement is of all or substantially all functionalmouse immunoglobulin heavy chain segments (i.e., V_(H), D_(H), and J_(H)segments) with one or more functional human light chain segments (i.e.,V_(L) and J_(L) segments). In one embodiment, the replacement is of allor substantially all functional mouse heavy chain V_(H), D_(H), andJ_(H) segments with all or substantially all human Vλ or Vκ segments andat least one Jλ or Jκ segment. In a specific embodiment, the replacementincludes all or substantially all functional human Jλ or Jκ segments.

In one aspect, a method is provided for making a mouse that expresses apolypeptide that comprises a sequence derived from a humanimmunoglobulin Vλ or Vκ and/or Jλ or Jκ segment fused with a mouse heavychain constant region, comprising replacing endogenous mouse heavy chainimmunoglobulin variable segments (V_(H), D_(H), and J_(H)) with at leastone human Vλ or Vκ segment and at least one human Jλ or Jκ segment,wherein the replacement is in a pluripotent, induced pluripotent, ortotipotent mouse cell to form a genetically modified mouse progenitorcell; the genetically modified mouse progenitor cell is introduced intoa mouse host; and, the mouse host comprising the genetically modifiedprogenitor cell is gestated to form a mouse comprising a genome derivedfrom the genetically modified mouse progenitor cell. In one embodiment,the host is an embryo. In a specific embodiment, the host is selectedfrom a mouse pre-morula (e.g., 8- or 4-cell stage), a tetraploid embryo,an aggregate of embryonic cells, or a blastocyst.

In one aspect, a method is provided for making a genetically modifiedmouse as described herein, comprising introducing by nuclear transfer anucleic acid containing a modification as described herein into a cell,and maintaining the cell under suitable conditions (e.g., includingculturing the cell and gestating an embryo comprising the cell in asurrogate mother) to develop into a mouse as described herein.

In one aspect, a method for making a modified mouse is provided,comprising modifying as described herein a mouse ES cell or pluripotentor totipotent or induced pluripotent mouse cell to include one or moreunrearranged immunoglobulin light chain variable gene segments operablylinked to an immunoglobulin heavy chain constant sequence, culturing theES cell, introducing the cultured ES cell into a host embryo to form achimeric embryo, and introducing the chimeric embryo into a suitablehost mouse to develop into a modified mouse. In one embodiment, the oneor more unrearranged immunoglobulin light chain variable region genesegments are human λ or human κ gene segments. In one embodiment, theone or more unrearranged immunoglobulin light chain variable region genesegments comprise human Vλ or human Vκ segments and one or more Jλ, Jκ,or J_(H) segments. In one embodiment, the heavy chain constant genesequence is a human sequence selected from C_(H)1, hinge, C_(H)2,C_(H)3, and a combination thereof. In one embodiment, the one or moreunrearranged immunoglobulin light chain variable gene segments replaceall or substantially all functional endogenous mouse heavy chainvariable region gene segments at the endogenous mouse heavy chain locus,and the heavy chain constant sequence is a mouse sequence comprising aC_(H)1, a hinge, a C_(H)2, and a C_(H)3.

In one aspect, an immunoglobulin variable region (VR) (e.g., comprisinga human V_(L) sequence fused with a human J_(L), or J_(H), or D_(H) andJ_(H), or D_(H) and J_(L)) made in a mouse as described herein isprovided. In a specific embodiment, the immunoglobulin VR is derivedfrom a germline human gene segment selected from a Vκ segment and a Vλsegment, wherein the VR is encoded by a rearranged sequence from themouse wherein the rearranged sequence is somatically hypermutated. Inone embodiment, the rearranged sequence comprises 1 to 5 somatichypermutations. In one embodiment, the rearranged sequence comprises atleast 6, 7, 8, 9, or 10 somatic hypermutations. In one embodiment, therearranged sequence comprises more than 10 somatic hypermutations. Inone embodiment, the rearranged sequence is fused with one or more humanor mouse heavy chain constant region sequences (e.g., selected from ahuman or mouse C_(H)1, hinge, C_(H)2, C_(H)3, and a combinationthereof).

In one aspect, an immunoglobulin variable domain amino acid sequence ofa binding protein made in a mouse as described herein is provided. Inone embodiment, the VR is fused with one or more human or mouse heavychain constant region sequences (e.g., selected from a human or mouseC_(H)1, hinge, C_(H)2, C_(H)3, and a combination thereof).

In one aspect, a light chain variable domain encoded by a nucleic acidsequence derived from a mouse as described herein is provided.

In one aspect, an antibody or antigen-binding fragment thereof (e.g.,Fab, F(ab)₂, scFv) made in a mouse as described herein, or derived froma sequence made in a mouse as described herein, is provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates a schematic (not to scale) of the mouse heavy chainlocus. The mouse heavy chain locus is about 3 Mb in length and containsapproximately 200 heavy chain variable (V_(H)) gene segments, 13 heavychain diversity (D_(H)) gene segments and 4 heavy chain joining (J_(H))gene segments as well as enhancers (Enh) and heavy chain constant(C_(H)) regions.

FIG. 1B illustrates a schematic (not to scale) of the human κ lightchain locus. The human κ light chain locus is duplicated into distal andproximal contigs of opposite polarity spanning about 440 kb and 600 kb,respectively. Between the two contigs is about 800 kb of DNA that isbelieved to be free of Vκ gene segments. The human κ light chain locuscontains about 76 Vκ gene segments, 5 Jκ gene segments, an intronicenhancer (Enh) and a single constant region (CIO.

FIG. 2 shows a targeting strategy for progressive insertion of 40 humanVκ and 5 human Jκ gene segments into the mouse heavy chain locus.Hygromycin (HYG) and Neomycin (NEO) selection cassettes are shown withrecombinase recognition sites (R1, R2, etc.).

FIG. 3 shows a modified mouse heavy chain locus comprising human Vκ andJκ gene segments operably linked to mouse C_(H) regions.

FIG. 4A shows an exemplary targeting strategy for progressive insertionof human Vλ and a single human Jλ gene segment into the mouse heavychain locus. Hygromycin (HYG) and Neomycin (NEO) selection cassettes areshown with recombinase recognition sites (R1, R2, etc.).

FIG. 4B shows an exemplary targeting strategy for progressive insertionof human Vλ and four human Jλ gene segments into the mouse heavy chainlocus. Hygromycin (HYG) and Neomycin (NEO) selection cassettes are shownwith recombinase recognition sites (R1, R2, etc.).

FIG. 5A shows an exemplary targeting strategy for progressive insertionof human Vλ human D_(H) and human J_(H) gene segments into the mouseheavy chain locus. Hygromycin (HYG) and Neomycin (NEO) selectioncassettes are shown with recombinase recognition sites (R1, R2, etc.).

FIG. 5B shows an exemplary targeting strategy for progressive insertionof human Vλ human D_(H) and human Jκ gene segments into the mouse heavychain locus. Hygromycin (HYG) and Neomycin (NEO) selection cassettes areshown with recombinase recognition sites (R1, R2, etc.).

FIG. 6A shows contour plots of splenocytes stained for surfaceexpression of B220 and IgM from a representative wild type (WT) and arepresentative mouse homozygous for six human Vκ and five human Jκ genesegments positioned at the endogenous heavy chain locus (6hVκ-5hJκ HO).

FIG. 6B shows contour plots of splenocytes gated on CD19⁺ B cells andstained for immunoglobulin D (IgD) and immunoglobulin M (IgM) from arepresentative wild type (WT) and a representative mouse homozygous forsix human Vκ and five human Jκ gene segments positioned at theendogenous heavy chain locus (6hVκ-5hJκ HO).

FIG. 6C shows the total number of CD19⁺ B cells, transitional B cells(CD19⁺IgM^(hi)IgD^(int)) and mature B cells (CD19⁺IgM^(int)IgD^(hi)) inharvested spleens from wild type (WT) and mice homozygous for six humanVκ and five human Jκ gene segments positioned at the endogenous heavychain locus (6hVκ-5hJκ HO).

FIG. 7A shows contour plots of bone marrow gated on singlets stained forimmunoglobulin M (IgM) and B220 from a wild type mouse (WT) and a mousehomozygous for six human Vκ and five human Jκ gene segments positionedat the endogenous heavy chain locus (6hVκ-5hJκ HO). Immature, mature andpro/pre B cells are noted on each of the dot plots.

FIG. 7B shows the total number of pre/pro (B220⁺IgM⁻), immature(B220^(int)IgM⁺) and mature (B220^(hi)IgM⁺) B cells in bone marrowisolated from the femurs of wild type mice (WT) and mice homozygous forsix human Vκ and five human Jκ gene segments positioned at theendogenous heavy chain locus (6hVκ-5hJκ HO).

FIG. 7C shows contour plots of bone marrow gated on CD19⁺ and stainedfor ckit⁺ and CD43⁺ from a wild type mouse (WT) and a mouse homozygousfor six human Vκ and five human Jκ gene segments positioned at theendogenous heavy chain locus (6hVκ-5hJκ HO). Pro and pre B cells arenoted on each of the dot plots.

FIG. 7D shows the number of pro B (CD19⁺CD43⁺ckit⁺) and pre B(CD19⁺CD43⁻ckit⁻) cells in bone marrow harvested from the femurs of wildtype mice (WT) and mice homozygous for six human Vκ and five human Jκgene segments positioned at the endogenous heavy chain locus (6hVκ-5hJκHO).

FIG. 7E shows contour plots of bone marrow gated on singlets stained forCD19 and CD43 from a wild type mouse (WT) and a mouse homozygous for sixhuman Vκ and five human Jκ gene segments positioned at the endogenousheavy chain locus (6hVκ-5hJκ HO). Immature, pre and pro B cells arenoted on each of the dot plots.

FIG. 7F shows histograms of bone marrow gated on pre B cells(CD19⁺CD43^(int)) and expressing immunoglobulin M (IgM) from a wild typemouse (WT) and a mouse homozygous for six human Vκ and five human Jκgene segments positioned at the endogenous heavy chain locus (6hVk-5hJkHO).

FIG. 7G shows the number of IgM⁺ pre B cells (CD19⁺IgM⁺CD43^(int)) andimmature B cells (CD19⁺IgM⁺CD43⁻) in bone marrow harvest from the femursof wild type (WT) and mice homozygous for six human Vκ and five human Jκgene segments positioned at the endogenous heavy chain locus (6hVκ-5hJκHO).

FIG. 8A shows contour plots of splenocytes gated on CD19⁺ and stainedfor Igλ⁺ and Igλ⁺ expression from a mouse containing a wild type heavychain locus and a replacement of the endogenous Vκ and Jκ gene segmentswith human Vκ and Jκ gene segments (WT) and a mouse homozygous forthirty hVκ and five Jκ gene segments at the endogenous heavy chain locusand a replacement of the endogenous Vκ and Jκ gene segments with humanVκ and Jκ gene segments (30hVκ-5hJκ HO).

FIG. 8B shows contour plots of bone marrow gated on immature(B220^(int)IgM⁺) and mature (B220^(hi)IgM⁺) B cells stained for Igλ andIgκ expression isolated from the femurs of a mouse containing a wildtype heavy chain locus and a replacement of the endogenous Vκ and Jκgene segments with human Vκ and Jκ gene segments (WT) and a mousehomozygous for thirty hVκ and five Jκ gene segments at the endogenousheavy chain locus and a replacement of the endogenous Vκ and Jκ genesegments with human Vκ and Jκ gene segments (30hVκ-5hJκ HO).

FIG. 9 shows a nucleotide sequence alignment of the Vκ-Jκ-mIgG junctionof twelve independent RT-PCR clones amplified from splenocyte RNA ofnaïve mice homozygous for thirty hVκ and five Jκ gene segments at themouse heavy chain locus and a replacement of the endogenous Vκ and Jκgene segments with human Vκ and Jκ gene segment. Lower case basesindicate non-germline bases resulting from either mutation and/or Naddition during recombination. Artificial spaces (periods) are includedto properly align the Framework 4 region and show alignment of the mouseheavy chain IgG nucleotide sequence for IgG1, IgG2a/c, and IgG3 primedclones.

DETAILED DESCRIPTION

The phrase “bispecific binding protein” includes a binding proteincapable of selectively binding two or more epitopes. Bispecific bindingproteins comprise two different polypeptides that comprise a first lightchain variable domain (V_(L)1) fused with a first C_(H) region and asecond light chain variable domain (V_(L)2) fused with a second C_(H)region. In general, the first and the second C_(H) regions areidentical, or they differ by one or more amino acid substitutions (e.g.,as described herein). V_(L)1 and V_(L)2 specifically binding differentepitopes—either on two different molecules (e.g., antigens) or on thesame molecule (e.g., on the same antigen). If a bispecific bindingprotein selectively binds two different epitopes (a first epitope and asecond epitope), the affinity of V_(L)1 for the first epitope willgenerally be at least one to two or three or four orders of magnitudelower than the affinity of V_(L)1 for the second epitope, and vice versawith respect to V_(L)2. The epitopes recognized by the bispecificbinding protein can be on the same or a different target (e.g., on thesame or a different antigen). Bispecific binding proteins can be made,for example, by combining a V_(L)1 and a V_(L)2 that recognize differentepitopes of the same antigen. For example, nucleic acid sequencesencoding V_(L) sequences that recognize different epitopes of the sameantigen can be fused to nucleic acid sequences encoding different C_(H)regions, and such sequences can be expressed in a cell that expresses animmunoglobulin light chain, or can be expressed in a cell that does notexpress an immunoglobulin light chain. A typical bispecific bindingprotein has two heavy chains each having three light chain CDRs,followed by (N-terminal to C-terminal) a C_(H)1 domain, a hinge, aC_(H)2 domain, and a C_(H)3 domain, and an immunoglobulin light chainthat either does not confer antigen-binding specificity but that canassociate with each heavy chain, or that can associate with each heavychain and that can bind one or more of the epitopes bound by V_(L)1and/or V_(L)2, or that can associate with each heavy chain and enablebinding or assist in binding of one or both of the heavy chains to oneor both epitopes.

Therefore, two general types of bispecific binding proteins are (1)V_(L)1-C_(H)(dimer), and (2) V_(L)1-C_(H):light chain+V_(L)2-C_(H):lightchain, wherein the light chain is the same or different. In either case,the C_(H) (i.e., the heavy chain constant region) can be differentiallymodified (e.g., to differentially bind protein A, to increase serumhalf-life, etc.) as described herein, or can be the same.

The term “cell,” when used in connection with expressing a sequence,includes any cell that is suitable for expressing a recombinant nucleicacid sequence. Cells include those of prokaryotes and eukaryotes(single-cell or multiple-cell), bacterial cells (e.g., strains of E.coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells,fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris,P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21,baculovirus-infected insect cells, Trichoplusia ni, etc.), non-humananimal cells, human cells, B cells, or cell fusions such as, forexample, hybridomas or quadromas. In some embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In some embodiments,the cell is eukaryotic and is selected from the following cells: CHO(e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell,Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK),HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21),Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell,SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myelomacell, tumor cell, and a cell line derived from an aforementioned cell.In some embodiments, the cell comprises one or more viral genes, e.g. aretinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

The term “cognate,” when used in the sense of “cognate with,” e.g., afirst V_(L) domain that is “cognate with” a second V_(L) domain, isintended to include reference to the relation between two V_(L) domainsfrom a same binding protein made by a mouse in accordance with theinvention. For example, a mouse that is genetically modified inaccordance with an embodiment of the invention, e.g., a mouse having aheavy chain locus in which V_(H), D_(H), and J_(H) regions are replacedwith V_(L) and J_(L) regions, makes antibody-like binding proteins thathave two identical polypeptide chains made of the same mouse C_(H)region (e.g., an IgG isotype) fused with a first human V_(L) domain, andtwo identical polypeptide chains made of the same mouse C_(L) regionfused with a second human V_(L) domain. During clonal selection in themouse, the first and the second human V_(L) domains were selected by theclonal selection process to appear together in the context of a singleantibody-like binding protein. Thus, first and second V_(L) domains thatappear together, as the result of the clonal selection process, in asingle antibody-like molecule are referred to as being “cognate.” Incontrast, a V_(L) domain that appears in a first antibody-like moleculeand a V_(L) domain that appears in a second antibody-like molecule arenot cognate, unless the first and the second antibody-like moleculeshave identical heavy chains (i.e., unless the V_(L) domain fused to thefirst human heavy chain region and the V_(L) domain fused to the secondhuman heavy chain region are identical).

The phrase “complementarity determining region,” or the term “CDR,”includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin genes that normally (i.e., in a wild-typeanimal) appears between two framework regions in a variable region of alight or a heavy chain of an immunoglobulin molecule (e.g., an antibodyor a T cell receptor). A CDR can be encoded by, for example, a germlinesequence or a rearranged or unrearranged sequence, and, for example, bya naïve or a mature B cell or a T cell. In some circumstances (e.g., fora CDR3), CDRs can be encoded by two or more sequences (e.g., germlinesequences) that are not contiguous (e.g., in an unrearranged nucleicacid sequence) but are contiguous in a B cell nucleic acid sequence,e.g., as the result of splicing or connecting the sequences (e.g., V-D-Jrecombination to form a heavy chain CDR3).

The phrase “gene segment,” or “segment” includes reference to a V (lightor heavy) or D or J (light or heavy) immunoglobulin gene segment, whichincludes unrearranged sequences at immunoglobulin loci (in e.g., humansand mice) that can participate in a rearrangement (mediated by, e.g.,endogenous recombinases) to form a rearranged V/J or V/D/J sequence.Unless indicated otherwise, the V, D, and J segments compriserecombination signal sequences (RSS) that allow for V/J recombination orV/D/J recombination according to the 12/23 rule. Unless indicatedotherwise, the segments further comprise sequences with which they areassociated in nature or functional equivalents thereof (e.g., for Vsegments promoter(s) and leader(s)).

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes animmunoglobulin heavy chain constant region sequence from any organism,and unless otherwise specified includes a heavy chain variable domain(V_(H)). V_(H) domains include three heavy chain CDRs and four framework(FR) regions, unless otherwise specified. Fragments of heavy chainsinclude CDRs, CDRs and FRs, and combinations thereof. A typical heavychain consists essentially of, following the variable domain (fromN-terminal to C-terminal), a C_(H)1 domain, a hinge, a C_(H)2 domain, aC_(H)3 domain, and optionally a C_(H)4 domain (e.g., in the case of IgMor IgE) and a transmembrane (M) domain (e.g., in the case ofmembrane-bound immunoglobulin on lymphocytes). A heavy chain constantregion is a region of a heavy chain that extends (from N-terminal sideto C-terminal side) from outside FR4 to the C-terminal of the heavychain. Heavy chain constant regions with minor deviations, e.g.,truncations of one, two, three or several amino acids from theC-terminal, would be encompassed by the phrase “heavy chain constantregion,” as well as heavy chain constant regions with sequencemodifications, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions. Amino acid substitutions can be made at one or morepositions selected from, e.g. (with reference to EU numbering of animmunoglobulin constant region, e.g., a human IgG constant region), 228,233, 234, 235, 236, 237, 238, 239, 241, 248, 249, 250, 252, 254, 255,256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286,289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308,309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331,332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360,361, 362, 373, 375, 376, 378, 380, 382, 383, 384, 386, 388, 389, 398,414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.

For example, and not by way of limitation, a heavy chain constant regioncan be modified to exhibit enhanced serum half-life (as compared withthe same heavy chain constant region without the recitedmodification(s)) and have a modification at position 250 (e.g., E or Q);250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S orT), and 256 (e.g., S/R/Q/E/D or T); or a modification at 428 and/or 433(e.g., L/R/SI/P/Q or K) and/or 434 (e.g., H/F or Y); or a modificationat 250 and/or 428; or a modification at 307 or 308 (e.g., 308F, V308F),and 434. In another example, the modification can comprise a 428L (e.g.,M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I),and a 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434(e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and256E) modification; a 250Q and 428L modification (e.g., T250Q andM428L); a 307 and/or 308 modification (e.g., 308F or 308P).

The phrase “light chain” includes an immunoglobulin light chain constant(C_(L)) region sequence from any organism, and unless otherwisespecified includes human κ and λ light chains. Light chain variable(V_(L)) domains typically include three light chain CDRs and fourframework (FR) regions, unless otherwise specified. Generally, afull-length light chain (V_(L)+C_(L)) includes, from amino terminus tocarboxyl terminus, a V_(L) domain that includesFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a C_(L) region. Light chains(V_(L)+C_(L)) that can be used with this invention include those, e.g.,that do not selectively bind either a first or second (in the case ofbispecific binding proteins) epitope selectively bound by the bindingprotein (e.g., the epitope(s) selectively bound by the V_(L) domainfused with the C_(H) domain). V_(L) domains that do not selectively bindthe epitope(s) bound by the V_(L) that is fused with the C_(H) domaininclude those that can be identified by screening for the most commonlyemployed light chains in existing antibody libraries (wet libraries orin silico), wherein the light chains do not substantially interfere withthe affinity and/or selectivity of the epitope binding domains of thebinding proteins. Suitable light chains include those that can bind(alone or in combination with its cognate V_(L) fused with the C_(H)region) an epitope that is specifically bound by the V_(L) fused to theC_(H) region.

The phrase “micromolar range” is intended to mean 1-999 micromolar; thephrase “nanomolar range” is intended to mean 1-999 nanomolar; the phrase“picomolar range” is intended to mean 1-999 picomolar.

The term “non-human animals” is intended to include any vertebrate suchas cyclostomes, bony fish, cartilaginous fish such as sharks and rays,amphibians, reptiles, mammals, and birds. Suitable non-human animalsinclude mammals. Suitable mammals include non-human primates, goats,sheep, pigs, dogs, cows, and rodents. Suitable non-human animals areselected from the rodent family including rat and mouse. In oneembodiment, the non-human animals are mice.

Mice, Nucleotide Sequences, and Binding Proteins

Binding proteins are provided that are encoded by elements ofimmunoglobulin loci, wherein the binding proteins compriseimmunoglobulin heavy chain constant regions fused with immunoglobulinlight chain variable domains. Further, multiple strategies are providedto genetically modify an immunoglobulin heavy chain locus in a mouse toencode binding proteins that contain elements encoded by immunoglobulinlight chain loci. Such genetically modified mice represent a source forgenerating unique populations of binding proteins that have animmunoglobulin structure, yet exhibit an enhanced diversity overtraditional antibodies.

Binding protein aspects described herein include binding proteins thatare encoded by modified immunoglobulin loci, which are modified suchthat gene segments that normally (i.e., in a wild-type animal) encodeimmunoglobulin light chain variable domains (or portions thereof) areoperably linked to nucleotide sequences that encode heavy chain constantregions. Upon rearrangement of the light chain gene segments, arearranged nucleotide sequence is obtained that comprises a sequenceencoding a light chain variable domain fused with a sequence encoding aheavy chain constant region. This sequence encodes a polypeptide thathas an immunoglobulin light chain variable domain fused with a heavychain constant region. Thus, in one embodiment, the polypeptide consistsessentially of, from N-terminal to C-terminal, a V_(L) domain, a C_(H)1region, a hinge, a C_(H)2 region, a C_(H)3 region, and optionally aC_(H)4 region.

In modified mice described herein, such binding proteins are made thatalso comprise a cognate light chain, wherein in one embodiment thecognate light chain pairs with the polypeptide described above to make abinding protein that is antibody-like, but the binding protein comprisesa V_(L) region—not a V_(H) region—fused to a C_(H) region.

In various embodiments, the modified mice make binding proteins thatcomprise a V_(L) region fused with a C_(H) region (a hybrid heavychain), wherein the V_(L) region of the hybrid heavy chain exhibits anenhanced degree of somatic hypermutation. In these embodiments, theenhancement is over a V_(L) region that is fused with a C_(L) region (alight chain). In some embodiments, a V_(L) region of a hybrid heavychain exhibits about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold,4-fold, 4.5-fold, or 5-fold or more somatic hypermutations than a V_(L)region fused with a C_(L) region. In some embodiments, the modified micein response to an antigen exhibit a population of binding proteins thatcomprise a V_(L) region of a hybrid heavy chain, wherein the populationof binding proteins exhibits an average of about 1.5-fold, 2-fold,2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold or more somatichypermutations in the V_(L) region of the hybrid heavy chain than isobserved in a wild-type mouse in response to the same antigen. In oneembodiment, the somatic hypermutations in the V_(L) region of the hybridheavy chain comprise one or more or two or more N additions in a CDR3.

In various embodiments, the binding proteins comprise variable domainsencoded by immunoglobulin light chain sequences that comprise a largernumber of N additions than observed in nature for light chainsrearranged from an endogenous light chain locus, e.g., a binding proteincomprising a mouse heavy chain constant region fused with a variabledomain derived from human light chain V gene segments and human (lightor heavy) J gene segments, wherein the human V and human J segmentsrearrange to form a rearranged gene that comprises 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more N additions.

In various embodiments, the mice of the invention make binding proteinsthat are on average smaller than wild-type antibodies (i.e., antibodiesthat have a V_(H) domain), and possess advantages associated withsmaller size. Smaller size is realized at least in part through theabsence of an amino acid sequence encoded by a D_(H) region, normallypresent in a V_(H) domain. Smaller size can also be realized in theformation of a CDR3 that is derived, e.g., from a Vκ region and a Jκregion.

In another aspect, a mouse and a method is provided for providing apopulation of binding proteins having somatically hypermutated V_(L)domains, e.g., somatically mutated human Vκ domains, and, e.g., human Vκdomains encoded by rearranged κ variable genes that comprise 1-10 ormore N additions. In one embodiment, in the absence of a V_(H) regionfor generating antibody diversity, a mouse of the invention willgenerate binding proteins, e.g., in response to challenge with anantigen, whose V domains are only or substantially V_(L) domains. Theclonal selection process of the mouse therefore is limited to selectingonly or substantially from binding proteins that have V_(L) domains,rather than V_(H) domains. Somatic hypermutation of the V_(L) domainswill be as frequent, or substantially more frequent (e.g., 2- to 5-foldhigher, or more), than in wild-type mice (which also mutate V_(L)domains with some frequency). The clonal selection process in a mouse ofthe invention will generate high affinity binding proteins from themodified immunoglobulin locus, including binding proteins thatspecifically bind an epitope with an affinity in the nanomolar orpicomolar range. Sequences that encode such binding proteins can be usedto make therapeutic binding proteins containing human variable regionsand human constant regions using an appropriate expression system.

In other embodiments, a mouse according to the invention can be madewherein the mouse heavy chain and/or light chain immunoglobulin loci aredisabled, rendered non-functional, or knocked out, and fully human orchimeric human-mouse transgenes can be placed in the mouse, wherein atleast one of the transgenes contains a modified heavy chain locus (e.g.,having light chain gene segments operably linked to one or more heavychain gene sequences). Such a mouse may also make a binding protein asdescribed herein.

In one aspect, a method is provided for increasing the diversity,including by somatic hypermutation or by N additions in a V_(L) domain,comprising placing an unrearranged light chain V gene segment and anunrearranged J gene segment in operable linkage with a mouse C_(H) genesequence, exposing the animal to an antigen of interest, and isolatingfrom the animal a rearranged and somatically hypermutated V(light)/Jgene sequence of the animal, wherein the rearranged V(light)/J genesequence is fused with a nucleotide sequence encoding an immunoglobulinC_(H) region.

In one embodiment, the immunoglobulin heavy chain fused with thehypermutated V_(L) is an IgM; in another embodiment, an IgG; in anotherembodiment, an IgE; in another embodiment, an IgA.

In one embodiment, the somatically hypermutated and class-switched V_(L)domain contains about 2- to 5-fold or more of the somatic hypermutationsobserved for a rearranged and class-switched antibody having a V_(L)sequence that is operably linked to a C_(L) sequence. In one embodiment,the observed somatic hypermutations in the somatically hypermutatedV_(L) domain are about the same in number as observed in a V_(H) domainexpressed from a V_(H) gene fused to a C_(H) region.

In one aspect, a method for making a high-affinity human V_(L) domain isprovided, comprising exposing a mouse of the invention to an antigen ofinterest, allowing the mouse to develop an immune response to theantigen of interest, and isolating a somatically mutated, class-switchedhuman V_(L) domain from the mouse that specifically binds the antigen ofinterest with high affinity.

In one embodiment, the K_(D) of a binding protein comprising thesomatically mutated, class-switched human V_(L) domain is in thenanomolar or picomolar range.

In one embodiment, the binding protein consists essentially of apolypeptide dimer, wherein the polypeptide consists essentially of thesomatically mutated, class-switched binding protein comprising a humanV_(L) domain fused with a human C_(H) region.

In one embodiment, the binding protein consists essentially of apolypeptide dimer and two light chains, wherein the polypeptide consistsessentially of the somatically mutated, class-switched binding proteinhaving a human V_(L) domain fused with a human C_(H) region; and whereineach polypeptide of the dimer is associated with a cognate light chaincomprising a cognate light chain V_(L) domain and a human C_(L) region.

In one aspect, a method is provided for somatically hypermutating ahuman V_(L) gene sequence, comprising placing a human V_(L) gene segmentand a human J_(L) gene segment in operable linkage with an endogenousmouse C_(H) gene at an endogenous mouse heavy chain immunoglobulinlocus, exposing the mouse to an antigen of interest, and obtaining fromthe mouse a somatically hypermutated human V_(L) gene sequence thatbinds the antigen of interest.

In one embodiment, the method further comprises obtaining from the mousea V_(L) gene sequence from a light chain that is cognate to the humansomatically hypermutated human V_(L) gene sequence that binds theantigen of interest.

V_(L) Binding Proteins with D_(H) Sequences

In various aspects, mice comprising an unrearranged immunoglobulin lightchain V gene segment and an unrearranged (e.g., light or heavy) J genesegment also comprise an unrearranged DH gene segment that is capable ofrecombining with the J segment to form a rearranged D/J sequence, whichin turn is capable of rearranging with the light chain V segment to forma rearranged variable sequence derived from (a) the light chain Vsegment, (b) the DH segment, and (c) the (e.g., light or heavy) Jsegment; wherein the rearranged variable sequence is operably linked toa heavy chain constant sequence (e.g., selected from CH1, hinge, CH2,CH3, and a combination thereof; e.g., operably linked to a mouse orhuman CH1, a hinge, a CH2, and a CH3).

In various aspects, mice comprising unrearranged human light chain Vsegments and J segments that also comprise a human D segment are useful,e.g., as a source of increased diversity of CDR3 sequences. Normally,CDR3 sequences arise in light chains from V/J recombination, and inheavy chains from V/D/J recombination. Further diversity is provided bynucleotide additions that occur during recombination (e.g., Nadditions), and also as the result of somatic hypermutation. Bindingcharacteristics conferred by CDR3 sequences are generally limited tothose conferred by the light chain CDR3 sequence, the heavy chain CDR3sequence, and a combination of the light and the heavy chain CDR3sequence, as the case may be. In mice as described herein, however, anadded source of diversity is available due to binding characteristicsconferred as the result of a combination of a first light chain CDR3 (onthe heavy chain polypeptide) and a second light chain CDR3 (on the lightchain polypeptide). Further diversity is possible where the first lightchain CDR3 may contain a sequence derived from a D gene segment, as froma mouse as described herein that comprises an unrearranged V segmentfrom a light chain V region operably linked to a D segment and operablylinked to a J segment (light or heavy), employing the RSS engineering astaught here.

Another source of diversity is the N and/or P additions that can occurin the V(light)/J or V(light)/D/J recombinations that are possible inmice as described. Thus, mice described herein not only provide adifferent source of diversity (light chain-light chain) but also afurther source of diversity due to the addition of, e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more N additions in a rearranged V(light)/J or arearranged V(light)/D/J gene in a mouse as described herein.

In various aspects the use of a D gene segment operably linked to a Jgene segment and a light chain V gene segment provides an enhanceddiversity. Operable linkage of a DH segment in this instance willrequire that that D segment is capable of recombining with the J segmentwith which it is recited. Thus, the D segment will require to havejuxtaposed a downstream RSS that is matched to the RSS juxtaposedupstream of the J segment such that the D segment and the J segment mayrearrange. Further, the D segment will require an appropriate RSSjuxtaposed upstream that is matched to the RSS juxtaposed downstream ofthe V segment such that the rearranged D/J segment and the V segment mayrearrange to form a gene encoding a variable domain.

An RSS, or a recombination signal sequence, comprises a conservednucleic acid heptamer sequence separated, by 12 base pairs (bp) or 23base pairs (bp) of unconserved sequence, from a conserved nucleic acidnonamer sequence. RSS's are used by recombinases to achieve joining ofimmunoglobulin gene segments during the rearrangement process followingthe 12/23 rule. According to the 12/23 rule, a gene segment juxtaposedwith an RSS having a 12 bp (unconserved) spacer rearranges with a genesegment juxtaposed with an RSS having a 23 bp (unconserved) spacer;i.e., rearrangements between gene segments each having an RSS with a 12bp spacer, or each having an RSS with a 23 bp spacer, are generally notobserved.

In the case of the λ light chain locus, variable gene segments (Vλ genesegments) are flanked downstream (with respect to the direction oftranscription of the V sequence) with an RSS having a 23-mer spacer, andjoining gene segments (Jλ gene segments) are flanked upstream (withrespect to the direction of transcription of the J sequence) with an RSShaving a 12-mer spacer. Thus, Vλ and Jλ segments are flanked with RSS'sthat are compatible under the 12/23 rule, and therefore are capable ofrecombine during rearrangement.

At the κ locus in a wild-type organism, however, each functional Vκsegment is flanked downstream with an RSS having a 12-mer spacer. Jκsegments, therefore, have 23-mer spaces juxtaposed on the upstream sideof the Jκ segment. At the heavy chain locus, V_(H) gene segments arejuxtaposed downstream with an RSS having a 23-mer spacer, followed byD_(H) segment juxtaposed upstream and downstream with a 12-mer spacer,and J_(H) segments each with a 23-mer segment juxtaposed on the upstreamside of the J_(H) segment. At the heavy chain locus, D/J recombinationoccurs first, mediated by the downstream D_(H) RSS with the 12-merspacer and the upstream J_(H) RSS with the 23-mer spacer, to yield anintermediate rearranged D-J sequence having an RSS juxtaposed on theupstream side that has an RSS with a 12-mer spacer. The rearranged D-Jsegment having the RSS with the 12-mer juxtaposed on the upstream sidethen rearranges with the V_(H) segment having the RSS with the 23-merjuxtaposed on its downstream side to form a rearranged V/D/J sequence.

In one embodiment, a Vλ segment is employed at the heavy chain locuswith a J gene segment that is a Jλ segment, wherein the Vλ segmentcomprises an RSS juxtaposed on the downstream side of the Vλ sequence,and the RSS comprises a 23-mer spacer, and the J segment is a Jλ segmentwith an RSS juxtaposed on its upstream side having a 12-mer spacer(e.g., as found in nature).

In one embodiment, a Vλ segment is employed at the heavy chain locuswith a J gene segment that is a Jκ or a J_(H) gene segment, wherein theVλ sequence has juxtaposed on its downstream side an RSS comprising a23-mer spacer, and the Jκ or J_(H) segment has juxtaposed on itsupstream side an RSS comprising a 12-mer spacer.

In one embodiment, a Vλ segment is employed at the heavy chain locuswith a D_(H) gene segment and a J gene segment. In one embodiment, theVλ segment comprises an RSS juxtaposed on the downstream side of the Vλsequence with an RSS having a 23-mer spacer; the D_(H) segment comprisesan RSS juxtaposed on the upstream side and on the downstream side of theD_(H) sequence with an RSS having a 12-mer spacer; and a J segmenthaving an RSS juxtaposed on its upstream side having a 23-mer spacer,wherein the J segment is selected from a Jλ, a Jκ, and a J_(H).

In one embodiment, a Vκ segment is employed at the heavy chain locuswith a J gene segment (with no intervening D segment), wherein the Vκsegment has an RSS juxtaposed on the downstream side of the Vκ segmentthat comprises a 12-mer spaced RSS, and the J segment has juxtaposed onits upstream side a 23-mer spaced RSS, and the Jκ segment is selectedfrom a Jκ segment, a Jλ segment, and a J_(H) segment. In one embodiment,the V segment and/or the J segment are human.

In one embodiment, the Vκ segment is employed at the heavy chain locuswith a D segment and a J segment, wherein the Vκ segment has an RSSjuxtaposed on the downstream side of the Vκ segment that comprises a12-mer spaced RSS, the D segment has juxtaposed on its upstream anddownstream side a 23-mer spaced RSS, and the J segment has juxtaposed onits upstream side a 12-mer spaced RSS. In one embodiment, the J segmentis selected from a Jκ segment, a Jλ segment, and a J_(H) segment. In oneembodiment, the V segment and/or the J segment are human.

A Jλ segment with an RSS having a 23-mer spacer juxtaposed at itsupstream end, or a Jκ or J_(H) segment with an RSS having a 12-merspacer juxtaposed at its upstream end, is made using any suitable methodfor making nucleic acid sequences that is known in the art. A suitablemethod for making a J segment having an RSS juxtaposed upstream whereinthe RSS has a selected spacer (e.g., either 12-mer or 23-mer) is tochemically synthesize a nucleic acid comprising the heptamer, thenonamer, and the selected spacer and fuse it to a J segment sequencethat is either chemically synthesized or cloned from a suitable source(e.g., a human sequence source), and employ the fused J segment sequenceand RSS in a targeting vector to target the RSS-J to a suitable site.

A D segment with a 23-mer spaced RSS juxtaposed upstream and downstreamcan be made by any method known in the art. One method compriseschemically synthesizing the upstream 23-mer RSS and D segment sequenceand the downstream 23-mer RSS, and placing the RSS-flanked D segment ina suitable vector. The vector may be directed to replace one or moremouse D segments with a human D segment with 12-mer RSS sequencesjuxtaposed on the upstream and downstream sides, or directed to beinserted into, e.g., a humanized locus at a position between a human Vsegment and a human or mouse J segment.

Suitable nonamers and heptamers for RSS construction are known in theart (e.g., see Janeway's Immunobiology, 7th ed., Murphy et al., (2008,Garland Science, Taylor & Francis Group, LLC) at page 148, FIG. 4.5,incorporated by reference). Suitable nonconserved spacer sequencesinclude, e.g., spacer sequences observed in RSS sequences at human ormouse immunoglobulin loci.

Bispecific-Binding Proteins

The binding proteins described herein, and nucleotide sequences encodingthem, can be used to make multispecific binding proteins, e.g.,bispecific binding proteins. In this aspect, a first polypeptideconsisting essentially of a first V_(L) domain fused with a C_(H) regioncan associate with a second polypeptide consisting essentially of asecond V_(L) domain fused with a C_(H) region. Where the first V_(L)domain and the second V_(L) domain specifically bind a differentepitope, a bispecific-binding molecule can be made using the two V_(L)domains. The C_(H) region can be the same or different. In oneembodiment, e.g., one of the C_(H) regions can be modified so as toeliminate a protein A binding determinant, whereas the other heavy chainconstant region is not so modified. This particular arrangementsimplifies isolation of the bispecific binding protein from, e.g., amixture of homodimers (e.g., homodimers of the first or the secondpolypeptides).

In one aspect, the methods and compositions described herein are used tomake bispecific-binding proteins. In this aspect, a first V_(L) that isfused to a C_(H) region and a second V_(L) that is fused to a C_(H)region are each independently cloned in frame with a human IgG sequenceof the same isotype (e.g., a human IgG1, IgG2, IgG3, or IgG4). The firstV_(L) specifically binds a first epitope, and the second V_(L)specifically binds a second epitope. The first and second epitopes maybe on different antigens, or on the same antigen.

In one embodiment, the IgG isotype of the C_(H) region fused to thefirst V_(L) and the IgG isotype of the C_(H) region fused to the secondV_(L) are the same isotype, but differ in that one IgG isotype comprisesat least one amino acid substitution. In one embodiment, the at leastone amino acid substitution renders the heavy chain bearing thesubstitution unable or substantially unable to bind protein A ascompared with the heavy chain that lacks the substitution.

In one embodiment, the first C_(H) region comprises a first C_(H)3domain of a human IgG selected from IgG1, IgG2, and IgG4; and the secondC_(H) region comprises a second C_(H)3 domain of a human IgG selectedfrom IgG1, IgG2, and IgG4, wherein the second C_(H)3 domain comprises amodification that reduces or eliminates binding of the second C_(H)3domain to protein A.

In one embodiment, the second C_(H)3 domain comprises a 435Rmodification, numbered according to the EU index of Kabat. In anotherembodiment, the second C_(H)3 domain further comprises a 436Fmodification, numbered according to the EU index of Kabat.

In one embodiment, the second C_(H)3 domain is that of a human IgG1 thatcomprises a modification selected from the group consisting of D356E,L358M, N384S, K392N, V397M, and V422I, numbered according to the EUindex of Kabat.

In one embodiment, the second C_(H)3 domain is that of a human IgG2 thatcomprises a modification selected from the group consisting of N384S,K392N, and V422I, numbered according to the EU index of Kabat.

In one embodiment, the second C_(H)3 domain is that of a human IgG4comprising a modification selected from the group consisting of Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I, numbered according to theEU index of Kabat.

In one embodiment, the binding protein comprises C_(H) regions havingone or more modifications as recited herein, wherein the constant regionof the binding protein is nonimmunogenic or substantially nonimmunogenicin a human. In a specific embodiment, the C_(H) regions comprise aminoacid sequences that do not present an immunogenic epitope in a human. Inanother specific embodiment, the binding protein comprises a C_(H)region that is not found in a wild-type human heavy chain, and the C_(H)region does not comprise a sequence that generates a T-cell epitope.

EXAMPLES

The following examples are provided so as to describe how to make anduse methods and compositions of the invention, and are not intended tolimit the scope of what the inventors regard as their invention. Unlessindicated otherwise, temperature is indicated in Celsius, and pressureis at or near atmospheric.

Example I Introduction of Light Chain Gene Segments into a Heavy ChainLocus

Various targeting constructs were made using VELOCIGENE® geneticengineering technology (see, e.g., U.S. Pat. No. 6,586,251 andValenzuela, D. M., Murphy, A. J., Frendewey, D., Gale, N. W.,Economides, A. N., Auerbach, W., Poueymirou, W. T., Adams, N. C., Rojas,J., Yasenchak, J., Chernomorsky, R., Boucher, M., Elsasser, A. L., Esau,L., Zheng, J., Griffiths, J. A., Wang, X., Su, H., Xue, Y., Dominguez,M. G., Noguera, I., Torres, R., Macdonald, L. E., Stewart, A. F.,DeChiara, T. M., Yancopoulos, G. D. (2003). High-throughput engineeringof the mouse genome coupled with high-resolution expression analysis.Nat Biotechnol 21, 652-659) to modify mouse genomic Bacterial ArtificialChromosome (BAC) libraries. Mouse BAC DNA was modified by homologousrecombination to inactivate the endogenous mouse heavy chain locusthrough targeted deletion of V_(H), D_(H) and J_(H) gene segments forthe ensuing insertion of unrearranged human germline κ light chain genesequences (top of FIG. 2).

Briefly, the mouse heavy chain locus was deleted in two successivetargeting events using recombinase-mediated recombination. The firsttargeting event included a targeting at the 5′ end of the mouse heavychain locus using a targeting vector comprising from 5′ to 3′ a 5′ mousehomology arm, a recombinase recognition site, a neomycin cassette and a3′ homology arm. The 5′ and 3′ homology arms contained sequence 5′ ofthe mouse heavy chain locus. The second targeting event included atargeting at the 3′ end of the mouse heavy chain locus in the region ofthe J_(H) gene segments using a second targeting vector that containedfrom 5′ to 3′ a 5′ mouse homology arm, a 5′ recombinase recognitionsite, a second recombinase recognition site, a hygromycin cassette, athird recombinase recognition site, and a 3′ mouse homology arm. The 5′and 3′ homology arms contained sequence flanking the mouse J_(H) genesegments and 5′ of the intronic enhancer and constant regions. PositiveES cells containing a modified heavy chain locus targeted with bothtargeting vectors (as described above) were confirmed by karyotyping.DNA was then isolated from the double-targeted ES cells and subjected totreatment with a recombinase thereby mediating the deletion of genomicDNA of the mouse heavy chain locus between the 5′ recombinaserecognition site in the first targeting vector and the 5′ recombinaserecognition site in the second targeting vector, leaving a singlerecombinase recognition site and the hygromycin cassette flanked by tworecombinase recognition sites (see top of FIG. 2). Thus a modified mouseheavy chain locus containing intact C_(H) genes was created forprogressively inserting human κ germline gene segments in a precisemanner using targeting vectors described below.

Four separate targeting vectors were engineered to progressively insert40 human Vκ gene segments and five human Jκ gene segments into theinactivated mouse heavy chain locus (described above) using standardmolecular techniques recognized in the art (FIG. 2). The human κ genesegments used for engineering the four targeting constructs arenaturally found in proximal contig of the germline human κ light chainlocus (FIG. 1B and Table 1).

A ˜110,499 bp human genomic fragment containing the first six human Vκgene segments and five human Jκ gene segments was engineered to containa PI-SceI site 431 bp downstream (3′) of the human Jκ5 gene segment.Another PI-SceI site was engineered at the 5′ end of a ˜7,852 bp genomicfragment containing the mouse heavy chain intronic enhancer, the IgMswitch region (Sμ) and the IgM gene of the mouse heavy chain locus. Thismouse fragment was used as a 3′ homology arm by ligation to the ˜110.5kb human fragment, which created a 3′ junction containing, from 5′ to3′, ˜110.5 kb of genomic sequence of the human κ light chain locuscontaining the first six consecutive Vκ gene segments and five Jκ genesegments, a PI-SceI site, ˜7,852 bp of mouse heavy chain sequencecontaining the mouse intronic enhancer, Sμ and the mouse IgM constantgene. Upstream (5′) from the human W1-6 gene segment was an additional3,710 bp of human κ sequence before the start of the 5′ mouse homologyarm, which contained 19,752 bp of mouse genomic DNA corresponding tosequence 5′ of the mouse heavy chain locus. Between the 5′ homology armand the beginning of the human κ sequence was a neomycin cassetteflanked by three recombinase recognition sites (see Targeting Vector 1,FIG. 2). The final targeting vector for the first insertion of human κsequence from 5′ to 3′ included a 5′ homology arm containing ˜20 kb ofmouse genomic sequence 5′ of the heavy chain locus, a first recombinaserecognition site (R1), a neomycin cassette, a second recombinaserecognition site (R2), a third recombinase recognition site (R3), ˜110.5kb of human genomic κ sequence containing the first six consecutivehuman Vκ gene segments and five human Jκ gene segments, a PI-SceI site,and a 3′ homology arm containing ˜8 kb of mouse genomic sequenceincluding the intronic enhancer, Sμ and the mouse IgM constant gene (seeFIG. 2, Targeting Vector 1). Homologous recombination with thistargeting vector created a modified mouse heavy chain locus containingsix human Vκ gene segments and five human Jκ gene segments operablylinked to the endogenous mouse heavy chain constant genes which, uponrecombination, leads to the formation of a hybrid heavy chain (i.e., ahuman Vκ domain and a mouse C_(H) region).

TABLE 1 Targeting Size of Human κ Gene Segments Added Vector Human κSequence Vκ Jκ 1 ~110.5 kb 4-1, 5-2, 7-3, 2-4, 1-5, 1-6 1-5 2 ~140 kb3-7, 1-8, 1-9, 2-10, 3-11, — 1-12, 1-13, 2-14, 3-15, 1-16 3 ~161 kb1-17, 2-18, 2-19, 3-20, 6-21, — 1-22, 1-23, 2-24, 3-25, 2-26, 1-27,2-28, 2-29, 2-30 4 ~90 kb 3-31, 1-32, 1-33, 3-34, 1-35, — 2-36, 1-37,2-38, 1-39, 2-40

Introduction of Ten Additional Human Vκ Gene Segments into a HybridHeavy Chain Locus.

A second targeting vector was engineered for introduction of 10additional human Vκ gene segments to the modified mouse heavy chainlocus described above (see FIG. 2, Targeting Vector 2). A 140,058 bphuman genomic fragment containing 12 consecutive human Vκ gene segmentsfrom the human κ light chain locus was engineered with a 5′ homology armcontaining mouse genomic sequence 5′ of the mouse heavy chain locus anda 3′ homology arm containing human genomic κ sequence. Upstream (5′)from the human Vκ1-16 gene segment was an additional 10,170 bp of humanκ sequence before the start of the 5′ mouse homology arm, which was thesame 5′ homology arm used for construction of Targeting Vector 1 (seeFIG. 2). Between the 5′ homology arm and the beginning of the human κsequence was a hygromycin cassette flanked by recombinase recognitionsites. The 3′ homology arm included a 31,165 bp overlap of human genomicκ sequence corresponding to the equivalent 5′ end of the ˜110.5 kbfragment of human genomic κ sequence of Targeting Vector 1 (FIG. 2). Thefinal targeting vector for the insertion of 10 additional human Vκ genesegments from 5′ to 3′ included a 5′ homology arm containing ˜20 kb ofmouse genomic sequence 5′ of the heavy chain locus, a first recombinaserecognition site (R1), a hygromycin cassette, a second recombinaserecognition site (R2) and ˜140 kb of human genomic κ sequence containing12 consecutive human Vλ gene segments, ˜31 kb of which overlaps with the5′ end of the human κ sequence of Targeting Vector 1 and serves as the3′ homology arm for this targeting construct. Homologous recombinationwith this targeting vector created a modified mouse heavy chain locuscontaining 16 human Vκ gene segments and five human Jκ gene segmentsoperably linked to the mouse heavy chain constant genes which, uponrecombination, leads to the formation of a hybrid heavy chain.

Introduction of Fourteen Additional Human Vκ Gene Segments into a HybridHeavy Chain Locus.

A third targeting vector was engineered for introduction of 14additional human Vκ gene segments to the modified mouse heavy chainlocus described above (see FIG. 2, Targeting Vector 3). A 160,579 bphuman genomic fragment containing 15 consecutive human Vκ gene segmentswas engineered with a 5′ homology arm containing mouse genomic sequence5′ of the mouse heavy chain locus and a 3′ homology arm containing humangenomic κ sequence. Upstream (5′) from the human Vκ2-30 gene segment wasan additional 14,687 bp of human κ sequence before the start of the 5′mouse homology arm, which was the same 5′ homology used for the previoustwo targeting vectors (described above, see also FIG. 2). Between the 5′homology arm and the beginning of the human κ sequence was a neomycincassette flanked by recombinase recognition sites. The 3′ homology armincluded a 21,275 bp overlap of human genomic κ sequence correspondingto the equivalent 5′ end of the ˜140 kb fragment of human genomic κsequence of Targeting Vector 2 (FIG. 2). The final targeting vector forthe insertion of 14 additional human Vκ gene segments from 5′ to 3′included a 5′ homology arm containing ˜20 kb of mouse genomic sequence5′ of the mouse heavy chain locus, a first recombinase recognition site(R1), a neomycin cassette, a second recombinase recognition site (R2)and ˜161 kb of human genomic κ sequence containing 15 human Vκ genesegments, ˜21 kb of which overlaps with the 5′ end of the human κsequence of Targeting Vector 2 and serves as the 3′ homology arm forthis targeting construct. Homologous recombination with this targetingvector created a modified mouse heavy chain locus containing 30 human Vκgene segments and five human Jκ gene segments operably linked to themouse heavy chain constant genes which, upon recombination, leads to theformation of a chimeric κ heavy chain.

Introduction of Ten Additional Human Vκ Gene Segments into a HybridHeavy Chain Locus.

A fourth targeting vector was engineered for introduction of 10additional human Vκ gene segments to the modified mouse heavy chainlocus described above (see FIG. 2, Targeting Vector 4). A 90,398 bphuman genomic fragment containing 16 consecutive human Vκ gene segmentswas engineered with a 5′ homology arm containing mouse genomic sequence5′ of the mouse heavy chain locus and a 3′ homology arm containing humangenomic κ sequence. Upstream (5′) from the human Vκ2-40 gene segment wasan additional 8,484 bp of human κ sequence before the start of the 5′mouse homology arm, which was the same 5′ homology as the previoustargeting vectors (described above, see also FIG. 2). Between the 5′homology arm and the beginning of the human κ sequence was a hygromycincassette flanked by recombinase recognition sites. The 3′ homology armincluded a 61,615 bp overlap of human genomic κ sequence correspondingto the equivalent 5′ end of the ˜160 kb fragment of human genomic κsequence of Targeting Vector 3 (FIG. 2). The final targeting vector forthe insertion of 10 additional human Vκ gene segments from 5′ to 3′included a 5′ homology arm containing ˜20 kb of mouse genomic sequence5′ of the mouse heavy chain locus, a first recombinase recognition site(R1), a hygromycin cassette, a second recombinase recognition site (R2)and −90 kb of human genomic κ sequence containing 16 human Vκ genesegments, ˜62 kb of which overlaps with the 5′ end of the human κsequence of Targeting Vector 3 and serves as the 3′ homology arm forthis targeting construct. Homologous recombination with this targetingvector created a modified mouse heavy chain locus containing 40 human Vκgene segments and five human Jκ gene segments operably linked to themouse heavy chain constant genes which, upon recombination, leads to theformation of a chimeric κ heavy chain (FIG. 3).

Using a similar approach as described above, other combinations of humanlight chain variable domains in the context of mouse heavy chainconstant regions are constructed. Additional light chain variabledomains may be derived from human Vλ and Jλ gene segments (FIGS. 4A and4B).

The human λ light chain locus extends over 1,000 kb and contains over 80genes that encode variable (V) or joining (J) segments. Among the 70 Vλgene segments of the human λ light chain locus, anywhere from 30-38appear to be functional gene segments according to published reports.The 70 Vλ sequences are arranged in three clusters, all of which containdifferent members of distinct V gene family groups (clusters A, B andC). Within the human λ light chain locus, over half of all observed Vλdomains are encoded by the gene segments 1-40, 1-44, 2-8, 2-14, and3-21. There are seven Jλ gene segments, only four of which are regardedas generally functional Jλ gene segments—Jλ1, Jλ2, Jλ3, and Jλ7. In somealleles, a fifth Jλ-Cλ gene segment pair is reportedly a pseudo gene(Cλ6). Incorporation of multiple human Jλ gene segments into a hybridheavy chain locus, as described herein, is constructed by de novosynthesis. In this way, a genomic fragment containing multiple human Jλgene segments in germline configuration is engineered with multiplehuman Vλ gene segments and allow for normal V-J recombination in thecontext of a heavy chain constant region.

Coupling light chain variable domains with heavy chain constant regionsrepresents a potentially rich source of diversity for generating uniqueV_(L) binding proteins with human V_(L) regions in non-human animals.Exploiting this diversity of the human λ light chain locus (or human κlocus as described above) in mice results in the engineering of uniquehybrid heavy chains and gives rise to another dimension of bindingproteins to the immune repertoire of genetically modified animals andtheir subsequent use as a next generation platform for the generation oftherapeutics.

Additionally, human D_(H) and J_(H) (or Jλ) gene segments can beincorporated with either human Vκ or Vλ gene segments to construct novelhybrid loci that will give rise, upon recombination, to novel engineeredvariable domains (FIGS. 5A and 5B). In this latter case, engineeringcombinations of gene segments that are not normally contained in asingle locus would require specific attention to the recombinationsignal sequences (RSS) that are associated with respective gene segmentssuch that normal recombination can be achieved when they are combinedinto a single locus. For example, V(D)J recombination is known to beguided by conserved noncoding DNA sequences, known as heptamer andnonamer sequences that are found adjacent to each gene segment at theprecise location at which recombination takes place. Between thesenoncoding DNA sequences are nonconserved spacer regions that either 12or 23 base pairs (bp) in length. Generally, recombination only occurs atgene segments located on the same chromosome and those gene segmentsflanked by a 12-bp spacer can be joined to a gene segment flanked by a23-bp spacer, i.e. the 12/23 rule, although joining two of D_(H) genesegments (each flanked by 12-bp spacers) has been observed in a smallproportion of antibodies. To allow for recombination between genesegments that do not normally have compatible spacers (e.g., Vκ and aD_(H) or D_(H) and Jλ), unique, compatible spacers are synthesized inadjacent locations with the desired gene segments for construction ofunique hybrid heavy chains that allow for successful recombination toform unique heavy chains containing light chain variable regions.

Thus, using the strategy outlined above for incorporation of human κlight chain gene segments into an endogenous heavy chain locus allowsfor the use of other combinations of human λ light chain gene segmentsas well as specific human heavy chain gene segments (e.g., D_(H) andJ_(H)) and combinations thereof.

Example II Identification of Targeted ES Cells Bearing Human Light ChainGene Segments at an Endogenous Heavy Chain Locus

The targeted BAC DNA made in the foregoing Examples was used toelectroporate mouse ES cells to created modified ES cells for generatingchimeric mice that express V_(L) binding proteins (i.e., human κ lightchain gene segments operably linked to mouse heavy chain constantregions). ES cells containing an insertion of unrearranged human κ lightchain gene segments were identified by the quantitative PCR assay,TAQMAN® (Lie and Petropoulos, 1998. Curr. Opin. Biotechnology 9:43-48).Specific primers sets and probes were design for insertion of human κsequences and associated selection cassettes, loss of mouse heavy chainsequences and retention of mouse sequences flanking the endogenous heavychain locus.

ES cells bearing the human κ light chain gene segments can betransfected with a construct that expresses a recombinase in order toremove any undesired selection cassette introduced by the insertion ofthe targeting construct containing human κ gene segments. Optionally,the selection cassette may be removed by breeding to mice that expressthe recombinase (e.g., U.S. Pat. No. 6,774,279). Optionally, theselection cassette is retained in the mice.

Example III Generation and Analysis of Mice Expressing V_(L) BindingProteins

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou, W. T., Auerbach, W.,Frendewey, D., Hickey, J. F., Escaravage, J. M., Esau, L., Dore, A. T.,Stevens, S., Adams, N. C., Dominguez, M. G., Gale, N. W., Yancopoulos,G. D., DeChiara, T. M., Valenzuela, D. M. (2007). F0 generation micefully derived from gene-targeted embryonic stem cells allowing immediatephenotypic analyses. Nat Biotechnol 25, 91-99). VELOCIMICE® (F0 micefully derived from the donor ES cell) independently bearing human κ genesegments at the mouse heavy chain locus were identified by genotypingusing a modification of allele assay (Valenzuela et al., supra) thatdetected the presence of the unique human κ gene segments at theendogenous heavy chain locus (supra). Pups are genotyped and a pupheterozygous for the hybrid heavy chain gene locus is selected forcharacterizing expression of V_(L) binding proteins.

Flow Cytometry.

The introduction of human κ light chain gene segments into the mouseheavy chain locus was carried out in an F1 ES line (F1H4; Valenzuela etal. 2007, supra) derived from 129S6/SvEvTac and C57BL/6NTac heterozygousembryos that further contained an in situ replacement of the mouse κlight chain gene segments with human κ light chain gene segments (U.S.Pat. No. 6,596,541). The human κ light chain germline variable genesegments are targeted to the 129S6 allele, which carries the IgM^(a)haplotype, whereas the unmodified mouse C576BL/6N allele bears theIgM^(b) haplotype. These allelic forms of IgM can be distinguished byflow cytometry using antibodies specific to the polymorphisms found inthe IgM^(a) or IgM^(b) alleles. Heterozygous mice bearing human κ lightchain gene segments at the endogenous heavy chain locus as described inExample I were evaluated for expression of human V_(L) binding proteinsusing flow cytometry.

Briefly, blood was drawn from groups of mice (n=6 per group) and grindedusing glass slides. C57BL/6 and Balb/c mice were used as control groups.Following lysis of red blood cells (RBCs) with ACK lysis buffer (LonzaWalkersville), cells were resuspended in BD Pharmingen FACS stainingbuffer and blocked with anti-mouse CD16/32 (BD Pharmingen). Lymphocyteswere stained with anti-mouse IgM^(b)-FITC (BD Pharmingen), anti-mouseIgM^(a)-PE (BD Pharmingen), anti-mouse CD19 (Clone 1D3; BD Biosciences),and anti-mouse CD3 (17A2; BIOLEGEND®) followed by fixation with BDCYTOFIX™ all according to the manufacturer's instructions. Final cellpellets were resuspended in staining buffer and analyzed using a BDFACSCALIBUR™ and BD CELLQUEST PRO™ software. Table 2 sets forth theaverage percent values for B cells (CD19⁺), T cells (CD3⁺), hybrid heavychain (CD19⁺IgM^(a+)), and wild type heavy chain (CD19⁺IgM^(b+))expression observed in groups of animals bearing each geneticmodification.

In a similar experiment, B cell contents of the spleen, blood and bonemarrow compartments from mice homozygous for six human Vκ and five humanJκ gene segments operably linked to the mouse heavy chain constantregion (described in Example I, FIG. 2) were analyzed for progressionthrough B cell development using flow cytometry of various cell surfacemarkers.

Briefly, two groups (n=3 each, 8 weeks old females) of wild type andmice homozygous for six human Vκ and five human Jκ gene segmentsoperably linked to the mouse heavy chain constant region were sacrificedand blood, spleens and bone marrow were harvested. Blood was collectedinto microtainer tubes with EDTA (BD Biosciences). Bone marrow wascollected from femurs by flushing with complete RPMI medium (RPMI mediumsupplemented with fetal calf serum, sodium pyruvate, Hepes,2-mercaptoethanol, non-essential amino acids, and gentamycin). RBCs fromspleen and bone marrow preparations were lysed with ACK lysis buffer(Lonza Walkersville), followed by washing with complete RPMI medium.

Cells (1×10⁶) were incubated with anti-mouse CD16/CD32 (2.4G2, BD) onice for ten minutes, followed by labeling with the following antibodycocktail for thirty minutes on ice: anti-mouse FITC-CD43 (1B11,BIOLEGEND®), PE-ckit (2B8, BIOLEGEND®), PeCy7-IgM (11/41, EBIOSCIENCE®),PerCP-Cy5.5-IgD (11-26c.2a, BIOLEGEND®), APC-eFluor 780-B220 (RA3-6B2,EBIOSCIENCE®), APC-CD19 (MB19-1, EBIOSCIENCE®). Bone marrow: immature Bcells (B220^(int)IgM⁺), mature B cells (B220^(hi)IgM⁺), pro B cells(CD19⁺ckit⁺CD43⁺), pre B cells (CD19⁺ckit⁻CD43⁻), pre-B cells(CD19⁺CD43^(int)IgM^(+/−)), immature B cells (CD19⁺CD43⁻IgM^(+/−)).Blood and spleen: B cells (CD19⁺), mature B cells(CD19⁺IgM^(int)IgD^(hi)), transitional/immature B cells(CD19⁺IgM^(hi)IgD^(int)).

Following staining, cells were washed and fixed in 2% formaldehyde. Dataacquisition was performed on a LSRII flow cytometer and analyzed withFLOWJO™ software (Tree Star, Inc.). FIGS. 6A, 6B and 6C show the resultsfor the splenic compartment. FIG. 7A-7G show the results for the bonemarrow compartment. The results obtained for the blood compartment fromeach group of mice demonstrated similar results as compared to theresults from the splenic compartment from each group (data not shown).

In a similar experiment, B cell contents of the spleen, blood and bonemarrow compartments from mice homozygous for thirty human Vκ and fivehuman Jκ gene segments operably linked to the mouse heavy chain constantregion (described in Example I, FIG. 2) were analyzed for progressionthrough B cell development using flow cytometry of various cell surfacemarkers.

Briefly, two groups (N=3 each, 6 week old females) of mice containing awild-type heavy chain locus and a replacement of the endogenous Vκ andJκ gene segments with human Vκ and Jκ gene segments (WT) and micehomozygous for thirty hVκ and five Jκ gene segments and a replacement ofthe endogenous Vκ and Jκ gene segments with human Vκ and Jκ genesegments (30hVκ-5hJκ HO) were sacrificed and spleens and bone marrowwere harvested. Bone marrow and splenocytes were prepared for stainingwith various cell surface markers (as described above).

Cells (1×10⁶) were incubated with anti-mouse CD16/CD32 (2.4G2, BDBiosciences) on ice for ten minutes, followed by labeling with bonemarrow or splenocyte panels for thirty minutes on ice. Bone marrowpanel: anti-mouse FITC-CD43 (1B11, BIOLEGEND®), PE-ckit (2B8,BIOLEGEND®), PeCy7-IgM (11/41, EBIOSCIENCE®), APC-CD19 (MB19-1,EBIOSCIENCE®). Bone marrow and spleen panel: anti-mouse FITC-Igκ (187.1BD Biosciences), PE-Igλ (RML-42, BIOLEGEND®), PeCy7-IgM (11/41,EBIOSCIENCE®), PerCP-Cy5.5-IgD (11-26c.2a, BIOLEGEND®), Pacific Blue-CD3(17A2, BIOLEGEND®), APC-B220 (RA3-6B2, EBIOSCIENCE®), APC-H7-CD19 (1D3,BD). Bone marrow: immature B cells (B220^(int)IgM⁺), mature B cells(B220^(hi)IgM⁺), pro B cells (CD19⁺ckit⁺CD43⁺), pre B cells(CD19+ckit−CD43−), immature Igx⁺ B cells (B220^(int)IgM⁺Igκ⁺Igλ⁻),immature Igλ⁺ B cells (B220^(int)IgM⁺Igκ⁻Igk⁺), mature Igκ⁺ B cells(B220^(hu)IgM⁺Igκ⁺Igλ⁻), mature IgX⁺ B cells (B220^(hi)IgM⁺Igκ⁻Igλ⁺).Spleen: B cells (CD19⁺), mature B cells (CD19⁺IgD^(hi)IgM^(int)),transitional/immature B cells (CD19⁺IgD^(int)IgM^(hi)). Bone marrow andspleen: Igx⁺ B cells (CD19⁺Igκ⁺Igλ⁻), Igλ⁺ B cells (CD19⁺Igκ⁻Igλ⁺).

Following staining, cells were washed and fixed in 2% formaldehyde. Dataacquisition was performed on a LSRII flow cytometer and analyzed withFLOWJO™ software (Tree Star, Inc.). The results demonstrated similarstaining patterns and cell populations for all three compartments ascompared to mice homozygous for six human Vκ and five human Jκ genesegments (described above). However, these mice demonstrated a loss inendogenous λ light chain expression in both the splenic and bone marrowcompartments (FIGS. 8A and 8B, respectively), despite the endogenouslight chain locus being intact in these mice. This may reflect aninability of rearranged human κ light chain domains, in the context ofheavy chain constant regions, to pair or associate with murine λ lightchain domains, leading to deletion of Igλ⁺ cells.

Isotype Expression.

Total and surface (i.e., membrane bound) immunoglobulin M (IgM) andimmunoglobulin G1 (IgG1) was determined for mice homozygous for humanheavy and κ light chain variable gene loci (VELCOIMMUNE® Humanized Mice,see U.S. Pat. No. 7,105,348) and mice homozygous for six human Vκ and 5human Jκ gene segments engineered into the endogenous heavy chain locus(6hVκ-5hJκ HO) by a quantitative PCR assay using TAQMAN® probes (asdescribed above in Example II).

Briefly, CD19⁺ B cells were purified from the spleens of groups of mice(n=3 to 4 mice per group) using mouse CD19 Microbeads (Miltenyi Biotec)according to manufacturer's instructions. Total RNA was purified usingthe RNEASY™ Mini kit (Qiagen). Genomic RNA was removed using anRNase-free DNase on-column treatment (Qiagen). About 200 ng mRNA wasreverse-transcribed into cDNA using the First Stand cDNA Synthesis kit(Invitrogen) and then amplified with the TAQMAN® Universal PCR MasterMix (Applied Biosystems) using the ABI 7900 Sequence Detection System(Applied Biosystems). Unique primer/probe combinations were employed tospecifically determine expression of total, surface (i.e.,transmembrane) and secreted forms of IgM and IgG1 isotypes (Table 3).Relative expression was normalized to the mouse κ constant region (mCκ).

TABLE 2 Mouse Genotype % CD3 % CD19 % IgM^(a) % IgM^(b) C57BL/6 22 63 0100 Balb/c 11 60 100 0 6hVκ-5hJκ HET 43 30 7 85 16hVκ-5hJκ HET 33 41 781

TABLE 3 SEQ ID Isotype Sequence (5′-3′) NOs: Surfacesense: GAGAGGACCG TGGACAAGTC 1 IgM antisense: TGACGGTGGT GCTGTAGAAG 2probe: ATGCTGAGGA GGAAGGCTTT GAGAACCT 3 Totalsense: GCTCGTGAGC AACTGAACCT 4 IgM antisense: GCCACTGCAC ACTGATGTC 5probe: AGTCAGCCAC AGTCACCTGC CTG 6 Surface sense: GCCTGCACAA CCACCATAC 7IgG1 antisense: GAGCAGGAAG AGGCTGATGA AG 8probe: AGAAGAGCCT CTCCCACTCT CCTGG 9 Totalsense: CAGCCAGCGG AGAACTACAA G 10 IgG1 antisense: GCCTCCCAGT TGCTCTTCTG11 probe: AACACTCAGC CCATCATGGA CACA 12 Cκ sense: TGAGCAGCAC CCTCACGTT13 antisense: GTGGCCTCAC AGGTATAGCT GTT 14 probe: ACCAAGGACG AGTATGAA 15

The results from the quantitative TAQMAN® PCR assay demonstrated adecrease in total IgM and total IgG1. However, the ratio of secretedversus surface forms of IgM and IgG1 appeared normal as compared toVELCOIMMUNE® humanized mice (data not shown).

Human κ Gene Segment Usage and Vκ-Jκ Junction Analysis.

Naïve mice homozygous for thirty hVκ and five Jκ gene segments and areplacement of the endogenous Vκ and Jκ gene segments with human Vκ andJκ gene segments (30hVκ-5hR HO) were analyzed for unique human Vκ-Jκrearrangements on mouse heavy chain (IgG) by reverse transcriptionpolymerase chain reaction (RT-PCR) using RNA isolated from splenocytes.

Briefly, spleens were harvested and perfused with 10 mL RPMI-1640(Sigma) with 5% HI-FBS in sterile disposable bags. Each bag containing asingle spleen was then placed into a STOMACHER™ (Seward) and homogenizedat a medium setting for 30 seconds. Homogenized spleens were filteredusing a 0.7 μm cell strainer and then pelleted with a centrifuge (1000rpm for 10 minutes) and RBCs were lysed in BD PHARM LYSE™ (BDBiosciences) for three minutes. Splenocytes were diluted with RPMI-1640and centrifuged again, followed by resuspension in 1 mL of PBS (IrvineScientific). RNA was isolated from pelleted splenocytes using standardtechniques known in the art.

RT-PCR was performed on splenocyte RNA using primers specific for humanhVκ gene segments and the mouse IgG. The mouse IgG primer was designedsuch that it was capable of amplifying RNA derived from all mouse IgGisotypes. PCR products were gel-purified and cloned into pCR2.1-TOPO TAvector (Invitrogen) and sequenced with primers M13 Forward (GTAAAACGACGGCCAG; SEQ ID NO:16) and M13 Reverse (CAGGAAACAG CTATGAC; SEQ ID NO:17)located within the vector at locations flanking the cloning site. HumanVκ and Jκ gene segment usage among twelve selected clones are shown inTable 4. FIG. 9 sets forth the nucleotide sequence of the hVκ-hJκ-mIgGjunction for the twelve selected RT-PCR clones.

As shown in this Example, mice homozygous for six human Vκ and fivehuman Jκ gene segments or homozygous for thirty human Vκ and five humanJκ gene segments operably linked to the mouse heavy chain constantregion demonstrated expression human light chain variable regions from amodified heavy chain locus containing light chain variable gene segmentsin their germline configuration. Progression through the various stagesof B cell development was observed in these mice, indicating multipleproductive recombination events involving light chain variable genesegments from an endogenous heavy chain locus and expression of suchhybrid heavy chains (i.e., human light chain variable region linked to aheavy chain constant region) as part of the antibody repertoire.

TABLE 4 Hybrid Heavy Chain Clone Vκ Jκ C_(H) SEQ ID NO: 1E 1-5  4IgG2A/C 18 1G 1-9  4 IgG2A/C 19 1A 1-16 5 IgG3 20 2E 1-12 2 IgG1 21 1C1-27 4 IgG2A/C 22 2H 2-28 1 IgG1 23 3D 3-11 4 IgG1 24 3A 3-20 4 IgG2A/C25 4B 4-1  5 IgG2A/C 26 4C 4-1  2 IgG3 27 5A 5-2  2 IgG2A/C 28 5D 5-2  1IgG1 29

Example IV Propagation of Mice Expressing V_(L) Binding Proteins

To create a new generation of V_(L) binding proteins, mice bearing theunrearranged human κ gene segments can be bred to another mousecontaining a deletion of the other endogenous heavy chain allele. Inthis manner, the progeny obtained would express only hybrid heavy chainsas described in Example I. Breeding is performed by standard techniquesrecognized in the art and, alternatively, by commercial companies, e.g.,The Jackson Laboratory. Mouse strains bearing a hybrid heavy chain locusare screened for presence of the unique hybrid heavy chains and absenceof traditional mouse heavy chains.

Alternatively, mice bearing the unrearranged human κ gene segments atthe mouse heavy chain locus can be optimized by breeding to other micecontaining one or more deletions in the mouse light chain loci (κ andλ). In this manner, the progeny obtained would express unique human κheavy chain only antibodies as described in Example I. Breeding issimilarly performed by standard techniques recognized in the art and,alternatively, by commercial companies, e.g., The Jackson Laboratory.Mouse strains bearing a hybrid heavy chain locus and one or moredeletions of the mouse light chain loci are screened for presence of theunique hybrid heavy chains containing human κ light chain domains andmouse heavy chain constant domains and absence of endogenous mouse lightchains.

Mice bearing an unrearranged hybrid heavy chain locus are also bred withmice that contain a replacement of the endogenous mouse κ light chainvariable gene locus with the human κ light chain variable gene locus(see U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, TheVELOCIMMUNE® Humanized Mouse Technology). The VELOCIMMUNE® HumanizedMouse includes, in part, having a genome comprising human κ light chainvariable regions operably linked to endogenous mouse κ light chainvariable constant region loci such that the mouse produces antibodiescomprising a human κ light chain variable domain and a mouse heavy chainconstant domain in response to antigenic stimulation. The DNA encodingthe variable regions of the light chains of the antibodies can beisolated and operably linked to DNA encoding the human light chainconstant regions. The DNA can then be expressed in a cell capable ofexpressing the fully human light chain of the antibody. Upon a suitablebreeding schedule, mice bearing a replacement of the endogenous mouse κlight chain with the human κ light chain locus and an unrearrangedhybrid heavy chain locus is obtained. Unique V_(L) binding proteinscontaining somatically mutated human Vκ domains can be isolated uponimmunization with an antigen of interest.

Example V Generation of V_(L) Binding Proteins

After breeding mice that contain the unrearranged hybrid heavy chainlocus to various desired strains containing modifications and deletionsof other endogenous Ig loci (as described in Example IV), selected micecan be immunized with an antigen of interest.

Generally, a VELOCIMMUNE® humanized mouse containing at least one hybridheavy chain locus is challenged with an antigen, and cells (such asB-cells) are recovered from the animal (e.g., from spleen or lymphnodes). The cells may be fused with a myeloma cell line to prepareimmortal hybridoma cell lines, and such hybridoma cell lines arescreened and selected to identify hybridoma cell lines that produceantibodies containing hybrid heavy chains specific to the antigen usedfor immunization. DNA encoding the human Vκ regions of the hybrid heavychains may be isolated and linked to desirable constant regions, e.g.,heavy chain and/or light chain. Due to the presence of human Vκ genesegments fused to the mouse heavy chain constant regions, a uniqueantibody-like repertoire is produced and the diversity of theimmunoglobulin repertoire is dramatically increased as a result of theunique antibody format created. This confers an added level of diversityto the antigen specific repertoire upon immunization. The resultingcloned antibody sequences may be subsequently produced in a cell, suchas a CHO cell. Alternatively, DNA encoding the antigen-specific V_(L)binding proteins or the variable domains may be isolated directly fromantigen-specific lymphocytes (e.g., B cells).

Initially, high affinity V_(L) binding proteins are isolated having ahuman Vκ region and a mouse constant region. As described above, theV_(L) binding proteins are characterized and selected for desirablecharacteristics, including affinity, selectivity, epitope, etc. Themouse constant regions are replaced with a desired human constant regionto generate unique fully human V_(L) binding proteins containingsomatically mutated human Vκ domains from an unrearranged hybrid heavychain locus of the invention. Suitable human constant regions include,for example wild type or modified IgG1 or IgG4 or, alternatively Cκ orCλ.

Separate cohorts of mice containing a replacement of the endogenousmouse heavy chain locus with six human Vκ and five human Jκ genesegments (as described in Example I) and a replacement of the endogenousVκ and Jκ gene segments with human Vκ and Jκ gene segments wereimmunized with a human cell-surface receptor protein (Antigen X).Antigen X is administered directly onto the hind footpad of mice withsix consecutive injections every 3-4 days. Two to three micrograms ofAntigen X are mixed with 10 μg of CpG oligonucleotide (Cat #tlrl-modn—ODN1826 oligonucleotide; InVivogen, San Diego, Calif.) and 25μg of Adju-Phos (Aluminum phosphate gel adjuvant, Cat# H-71639-250;Brenntag Biosector, Frederikssund, Denmark) prior to injection. A totalof six injections are given prior to the final antigen recall, which isgiven 3-5 days prior to sacrifice. Bleeds after the 4th and 6thinjection are collected and the antibody immune response is monitored bya standard antigen-specific immunoassay.

When a desired immune response is achieved splenocytes are harvested andfused with mouse myeloma cells to preserve their viability and formhybridoma cell lines. The hybridoma cell lines are screened and selectedto identify cell lines that produce Antigen X-specific V_(L) bindingproteins. Using this technique several anti-Antigen X-specific V_(L)binding proteins (i.e., binding proteins possessing human Vκ domains inthe context of mouse heavy and light chain constant domains) areobtained.

Alternatively, anti-Antigen X V_(L) binding proteins are isolateddirectly from antigen-positive B cells without fusion to myeloma cells,as described in U.S. 2007/0280945A1, herein specifically incorporated byreference in its entirety. Using this method, several fully humananti-Antigen X V_(L) binding proteins (i.e., antibodies possessing humanVκ domains and human constant domains) were obtained.

Human λ Gene Segment Usage.

To analyze the structure of the anti-Antigen X V_(L) binding proteinsproduced, nucleic acids encoding the human Vκ domains (from both theheavy and light chains of the V_(L) binding protein) were cloned andsequenced using methods adapted from those described in US2007/0280945A1 (supra). From the nucleic acid sequences and predictedamino acid sequences of the antibodies, gene usage was identified forthe hybrid heavy chain variable region of selected V_(L) bindingproteins obtained from immunized mice (described above). Table 5 setsfor the gene usage of human Vκ and Jκ gene segments from selectedanti-Antigen X V_(L) binding proteins, which demonstrates that miceaccording to the invention generate antigen-specific V_(L) bindingproteins from a variety of human Vκ and Jκ gene segments, due to avariety of rearrangements at the endogenous heavy chain and κ lightchain loci both containing unrearranged human Vκ and Jκ gene segments.Human Vκ gene segments rearranged with a variety of human Jκ segments toyield unique antigen-specific V_(L) binding proteins.

TABLE 5 Hybrid Heavy Chain Light Chain Antibody Vκ Jκ Vκ Jκ A 4-1 3 3-201 B 4-1 3 3-20 1 C 4-1 3 3-20 1 D 4-1 3 3-20 1 E 4-1 3 3-20 1 F 4-1 33-20 1 G 4-1 3 3-20 1 H 4-1 3 3-20 1 I 4-1 3 3-20 1 J 1-5 3 1-33 3 K 4-13 3-20 1 L 4-1 3 1-9  3 M 4-1 1 1-33 4 N 4-1 1 1-33 3 O 1-5 1 1-9  2 P1-5 3 1-16 4 Q 4-1 3 3-20 1 R 4-1 3 3-20 1 S 1-5 1 1-9  2 T 1-5 1 1-9  2U 5-2 2 1-9  3 V 1-5 2 1-9  2 W 4-1 1 1-33 4

Enzyme-Linked Immunosorbent Assay (ELISA).

Human V_(L) binding proteins raised against Antigen X were tested fortheir ability to block binding of Antigen X's natural ligand (Ligand Y)in an ELISA assay.

Briefly, Ligand Y was coated onto 96-well plates at a concentration of 2μg/mL diluted in PBS and incubated overnight followed by washing fourtimes in PBS with 0.05% Tween-20. The plate was then blocked with PBS(Irvine Scientific, Santa Ana, Calif.) containing 0.5% (w/v) BSA(Sigma-Aldrich Corp., St. Louis, Mo.) for one hour at room temperature.In a separate plate, supernatants containing anti-Antigen X V_(L)binding proteins were diluted 1:10 in buffer. A mock supernatant withthe same components of the V_(L) binding proteins was used as a negativecontrol. The extracellular domain (ECD) of Antigen X was conjugated tothe Fc portion of mouse IgG2a (Antigen X-mFc). Antigen X-mFc was addedto a final concentration of 0.150 nM and incubated for one hour at roomtemperature. The V_(L) binding protein/Antigen X-mFc mixture was thenadded to the plate containing Ligand Y and incubated for one hour atroom temperature. Detection of Antigen X-mFc bound to Ligand Y wasdetermined with Horse-Radish Peroxidase (HRP) conjugated toanti-Penta-His antibody (Qiagen, Valencia, Calif.) and developed bystandard colorimetric response using tetramethylbenzidine (TMB)substrate (BD Biosciences, San Jose, Calif.) neutralized by sulfuricacid. Absorbance was read at OD450 for 0.1 sec. Background absorbance ofa sample without Antigen X was subtracted from all samples. Percentblocking was calculated for >250 (three 96 well plates) AntigenX-specific V_(L) binding proteins by division of thebackground-subtracted MFI of each sample by the adjusted negativecontrol value, multiplying by 100 and subtracting the resulting valuefrom 100.

The results showed that several V_(L) binding proteins isolated frommice immunized with Antigen X specifically bound the extracellulardomain of Antigen X fused to the Fc portion of mouse IgG2a (data notshown).

Affinity Determination.

Equilibrium dissociation constants (K_(D)) for selected AntigenX-specific V_(L) binding protein supernatants were determined by SPR(Surface Plasmon Resonance) using a BIACORE™ T100 instrument (GEHealthcare). All data were obtained using HBS-EP (10 mM HEPES, 150 mMNaCl, 0.3 mM EDTA, 0.05% Surfactant P20, pH 7.4) as both the running andsample buffers, at 25° C.

Briefly, V_(L) binding proteins were captured from crude supernatantsamples on a CM5 sensor chip surface previously derivatized with a highdensity of anti-human Fc antibodies using standard amine couplingchemistry. During the capture step, supernatants were injected acrossthe anti-human Fc surface at a flow rate of 3 μL/min, for a total of 3minutes. The capture step was followed by an injection of either runningbuffer or analyte at a concentration of 100 nM for 2 minutes at a flowrate of 35 μL/min. Dissociation of antigen from the captured V_(L)binding protein was monitored for 6 minutes. The captured V_(L) bindingprotein was removed by a brief injection of 10 mM glycine, pH 1.5. Allsensorgrams were double referenced by subtracting sensorgrams frombuffer injections from the analyte sensorgrams, thereby removingartifacts caused by dissociation of the V_(L) binding protein from thecapture surface. Binding data for each V_(L) binding protein was fit toa 1:1 binding model with mass transport using BIAcore T100 Evaluationsoftware v2.1.

The binding affinities of thirty-four selected V_(L) binding proteinsvaried, with all exhibiting a K_(D) in the nanomolar range (1.5 to 130nM). Further, about 70% of the selected V_(L) binding proteins (23 of34) demonstrated single digit nanomolar affinity. T¹¹² measurements forthese selected V_(L) binding proteins demonstrated a range of about 0.2to 66 minutes. Of the thirty-four V_(L) binding proteins, six showedgreater than 3 nM affinity for Antigen X (1.53, 2.23, 2.58, 2.59, 2.79,and 2.84). The affinity data is consistent with the V_(L) bindingproteins resulting from the combinatorial association of rearrangedhuman light chain variable domains linked to heavy and light chainconstant regions (described in Table 4) being high-affinity, clonallyselected, and somatically mutated. The V_(L) binding proteins generatedby the mice described herein comprise a collection of diverse,high-affinity unique binding proteins that exhibit specificity for oneor more epitopes on Antigen X.

In another experiment, selected human V_(L) binding proteins raisedagainst Antigen X were tested for their ability to block binding ofAntigen X's natural ligand (Ligand Y) to Antigen X in a LUMINEX®bead-based assay (data not shown). The results demonstrated that inaddition to specifically binding the extracellular domain of Antigen Xwith affinities in the nanomolar range (described above), selected V_(L)binding proteins were also capable of binding Antigen X from cynomolgusmonkey (Macaca fascicularis).

1.-21. (canceled)
 22. A mouse whose genome comprises (a) a sequence thatincludes an unrearranged human light chain variable (V_(L)) gene segmentand an unrearranged human light chain joining (J_(L)) gene segmentoperably linked with a heavy chain constant (C_(H)) region; and (b) asequence that includes an unrearranged human light chain variable(V_(L)) gene segment and an unrearranged human light chain joining(J_(L)) gene segment operably linked with a light chain constant (C_(L))gene, wherein the unrearranged human V_(L) and J_(L) segments eachcomprise recombination signal sequences that allow for recombination toform a rearranged human V_(L)/J_(L) region, so that when immunized withan antigen, the mouse generates antibodies composed of two heavy chainsand two light chains, wherein: each heavy chain includes human V_(L) andJ_(L) amino acid sequences linked to a C_(H) amino acid sequence, eachlight chain includes human V_(L) and J_(L) amino acid sequences linkedto a C_(L) amino acid sequence.
 23. The mouse of claim 22(b), whereinthe C_(L) gene a mouse C_(L) gene.
 24. The mouse of claim 23, whereinthe mouse C_(L) gene is mouse Cκ gene.
 25. The mouse of claim 22(b),wherein the unrearranged human V_(L) and J_(L) gene segments are humanVκ and Jκ gene segments.
 26. The mouse of claim 22(a), wherein theunrearranged human V_(L) and J_(L) gene segments are human Vκ and Jκgene segments.
 27. The mouse of claim 22(a), wherein the unrearrangedhuman V_(L) and J_(L) gene segments are human Vλ and Jλ gene segments.28. The mouse of claim 22(a), wherein the sequence replaces all of theendogenous V_(H), D_(H) and J_(H) segments at the endogenous heavy chainlocus.
 29. The mouse of claim 22(a), wherein the sequence comprises aplurality of unrearranged human V_(L) gene segments and a plurality ofunrearranged human J_(L) gene segments; and wherein the C_(H) region isan endogenous C_(H) region.
 30. The mouse of claim 29, wherein thesequence includes (a) six human Vκ gene segments and five human Jκ genesegments; (b) 16 human Vκ gene segments and five human Jκ gene segments;(c) 30 human Vκ gene segments and five human Jκ gene segments; or (d) 40human Vκ gene segments and five human Jκ gene segments.
 31. The mouse ofclaim 29, wherein the sequence includes (a) 12 human Vλ gene segmentsand one human Jλ gene segment; (b) 28 human Vλ gene segments and onehuman Jλ gene segment; or (c) 40 human Vλ gene segments and one human Jλgene segment.
 32. The mouse of claim 31, wherein the one human Jλ genesegment is human Jλ1.
 33. The mouse of claim 29, wherein the sequenceincludes (a) 12 human Vλ gene segments and four human Jλ gene segments;(b) 28 human Vλ gene segments and four human Jλ gene segments; or (c) 40human Vλ gene segments and four human Jλ gene segments.
 34. The mouse ofclaim 33, wherein the four human Jλ gene segments are Jλ1, Jλ2, Jλ3 andJλ7.
 35. The mouse of claim 22(b), wherein the sequence comprises aplurality of unrearranged human V_(L) gene segments and a plurality ofunrearranged human J_(L) gene segments; and wherein the C_(L) region isan endogenous C_(L) region.
 36. An isolated mouse cell whose genomecomprises (a) a sequence that includes an unrearranged human V_(L) genesegment and an unrearranged human J_(L) gene segment operably linkedwith a C_(H) region; and (b) a sequence that includes an unrearrangedhuman V_(L) gene segment and an unrearranged human J_(L) gene segmentoperably linked with a light chain constant C_(L) gene, wherein theunrearranged human V_(L) and J_(L) segments each comprise recombinationsignal sequences that allow for recombination to form a rearranged humanV_(L)/J_(L) region.
 37. The isolated mouse cell of claim 36, wherein thecell is an embryonic stem (ES) cell.
 38. The isolated mouse cell ofclaim 36(a), wherein the sequence includes a plurality of unrearrangedhuman V_(L) gene segments and a plurality of unrearranged human J_(L)gene segments; and wherein the C_(H) region is an endogenous C_(H)region.
 39. The isolated mouse cell of claim 38, wherein theunrearranged human V_(L) and J_(L) gene segments are human Vκ and Jκgene segments
 40. The isolated mouse cell of claim 36(b), wherein thesequence includes a plurality of unrearranged human V_(L) gene segmentsand a plurality of unrearranged human J_(L) gene segments; and whereinthe C_(L) region is an endogenous C_(L) region.
 41. The isolated mousecell of claim 40, wherein the unrearranged human V_(L) and J_(L) genesegments are human Vκ and Jκ gene segments.